Expression Patterns of Neural Genes in Euperipatoides Kanangrensis Suggest Divergent Evolution of Onychophoran and Euarthropod Neurogenesis

Expression patterns of neural genes in Euperipatoides kanangrensis suggest divergent evolution of onychophoran and euarthropod neurogenesis

Bo Joakim Eriksson and Angelika Stollewerk1

School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS, United Kingdom

Edited by Thomas C. Kaufman, Indiana University, Bloomington, IN, and approved November 10, 2010 (received for review June 28, 2010) One of the controversial debates on euarthropod relationships pattern of neurogenesis that has been retained in these groups centers on the question as to whether insects, crustaceans, and and thus cannot be used to resolve euarthropod phylogeny? myriapods (Mandibulata) share a common ancestor or whether Analysis of neurogenesis in a closely related group, the Ony- myriapods group with the chelicerates (Myriochelata). The debate chophora, might shed light on this problem. Although the phy- was stimulated recently by studies in chelicerates and myriapods logenetic position of onychophorans is still debated, many that show that neural precursor groups (NPGs) segregate from the phylogenies group them with euarthropods and possibly tardi- neuroectoderm generating the nervous system, whereas in insects grades in the phylum Arthropoda; thus onychophorans share and crustaceans the nervous tissue is produced by stem cells. Do a common ancestor with euarthropods (8, 19–23). Analyses of the shared neural characters of myriapods and chelicerates repre- neurogenesis in onychophorans suggests that, similar to insects sent derived characters that support the Myriochelata grouping? and crustaceans, single neural precursors are formed in the neu- Or do they rather reflect the ancestral pattern? Analyses of neuro- roectoderm, rather than groups of cells as seen in chelicerates and genesis in a group closely related to euarthropods, the onycho- myriapods (24–26). This suggests that the nervous system of the phorans, show that, similar to insects and crustaceans, single neural last common ancestor of arthropods was generated by the seg- precursors are formed in the neuroectoderm, potentially support- regation of single neural precursor cells and thus the formation of ing the Myriochelata hypothesis. Here we show that the nature and single neuroblasts in insects and crustaceans would represent an EVOLUTION the selection of onychophoran neural precursors are distinct from ancestral character of neurogenesis. Consequently, the neural euarthropods. The onychophoran nervous system is generated by precursor groups (NPGs) of chelicerates and myriapods would be the massive irregular segregation of single neural precursors, a synapomorphy supporting the Myriochelata hypothesis and contrasting with the limited number and stereotyped arrangement contradicting the Mandibulata grouping (25). However, without of NPGs/stem cells in euarthropods. Furthermore, neural genes do a detailed analysis of the nature of the neural precursors in ony- not show the spatiotemporal pattern that sets up the precise chophorans, it is not possible to decide whether the similarities position of neural precursors as in euarthropods. We conclude that are superficial. fl neurogenesis in onychophorans largely does not re ect the ances- Here we analyze the morphological and molecular processes tral pattern of euarthropod neurogenesis, but shows a mixture of of neural precursor formation in the ventral neuroectoderm of derived characters and ancestral characters that have been modi- fi the onychophoran Euperipatoides kanangrensis, and compare our ed in the euarthropod lineage. Based on these data and additional results to the same processes in euarthropods. The formation of evidence, we suggest an evolutionary sequence of arthropod neu- neural precursors has been studied in all four arthropod groups; rogenesis that is in line with the Mandibulata hypothesis. however, molecular data are largely missing in crustaceans (10, 11, 27, 28). In insects, the stem cell-like neuroblasts segregate (de- achaete-scute homologue | euarthropod phylogeny | Notch signaling laminate) from a single layered neuroectoderm to the interior of the embryo in several phases. In this basal position, they divide here is an almost general agreement that (i) euarthropods asymmetrically to self-renew and to produce smaller ganglion Tderive from a common ancestor and (ii) crustaceans and mother cells that divide once to give rise to two neural cells. Ap- insects are sister groups, called Pancrustacea or Tetraconata (1–3). proximately 500 neuroblasts are generated in the ventral neuro- However, some issues of euarthropod relationships are still ectoderm, forming a highly stereotyped temporal and spatial controversial; for example the question as to whether insects, pattern. The cells remaining in the apical cell layer give rise to crustaceans, and myriapods form a monophyletic group, the epidermal cells (28). It has been shown in Drosophila melanogaster Mandibulata, or whether myriapods group with the chelicerates that the decision between epidermal and neural fate depends on to form the Myriochelata (4–9). Most phylogenies support the direct cell–cell interactions of the ventral neuroectodermal cells. Mandibulata, however, and evidence is based both on molecular The proneural genes achaete, scute, and lethal of scute are initially and morphological characters. In contrast, there are only few expressed in small clusters of neuroectodermal cells that consist of molecular phylogenies that favor the Myriochelata, and there- five to seven cells (29). Subsequently, their expression becomes fore this hypothesis is not widely accepted (4, 8, 9). The debate has been stimulated recently by morphological studies on the development of the nervous system that revealed a surprising Author contributions: A.S. designed research; B.J.E. performed research; A.S. contributed degree of similarity between myriapods and chelicerates (10, 11). new reagents/analytic tools; B.J.E. and A.S. analyzed data; and A.S. wrote the paper. While in insects and higher crustaceans (malacostracans) the The authors declare no conflict of interest. nervous system is generated by single stem cell-like cells (neu- This article is a PNAS Direct Submission. roblasts) (12, 13), in chelicerates and myriapods groups of neural Data deposition: The sequences reported in this paper have been deposited in the Gen- precursors are specified for the neural fate, which directly dif- Bank database (accession nos. EkASH, GU954550; EkDelta, GU954551; and EkNotch, GU954552). ferentiate into neural cells (10, 11, 14–18). However, do the 1To whom correspondence may be addressed. E-mail: [email protected] or a.stollewerk@ shared neural characters of myriapods and chelicerates represent qmul.ac.uk. synapomorphies (shared derived characters) that can support the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Myriochelata grouping? Or do they rather reflect the ancestral 1073/pnas.1008822108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1008822108 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 restricted to a single cell, the future neuroblast, by activation of Notch signaling in the remaining cells of the cluster (30). The expression of the proneural genes is highly dynamic and correlates with the position of neuroblast formation. Neuroblasts have also been detected in crustaceans, although most of the studies were done in representatives of higher crustaceans, the malacostracans (13, 31). Cell lineage studies have shown that malcostracan neu- roblasts divide in a stem cell-like manner, producing ganglion mother cells that divide only once to give rise to two neural cells, similar to the case in insects. Partial maps of crustacean neuro- blasts suggest a stereotyped arrangement of these cells, which again is similar to that in insects. Two achaete-scute homologs have Fig. 1. (A–E) Formation of neuromeres in the E. kanangrensis embryo. Light been identified in a single crustacean, the branchiopod Triops micrographs of transverse sections through the ventral neuroectoderm; longicaudatus, and their expression seems to correlate with neural dorsal is toward the top. Asterisks (*) label neuroecodermal cells. Some of precursor formation; however, a detailed study on the spatial and the segregating neural precursors are outlined in black. (A) Stage II, single temporal expression is missing (27). Interestingly, despite the dif- neural precursors (arrows) delaminate from the neuroectoderm (*) and form ference in neural precursor formation, the genetic network con- a loose basal layer between the outer ectoderm and the inner mesoderm. trolling this process is conserved in insects, chelicerates, and Before delamination, the neural precursors assume a bottle-like shape myriapods (10, 11, 14, 15, 32). However, the expression pattern and (arrowheads). (B) Neural precursors divide to generate smaller intermediate neural precursors (arrows). (C) Due to the segregation of additional neural function of these genes are adapted to the distinct morphology of precursors, the basal layer increases (arrows). (D) At stage III, the neuropile is neural precursor formation in the latter groups. In chelicerates and visible surrounded by differentiated neurons (large arrowheads). Small myriapods, the proneural genes are expressed in large domains arrowheads point to intermediate neural precursors; arrow indicates a seg- from which several NPGs segregate and the expression becomes regated precursor. (E) At stage IV, the basal area of differentiating neurons restricted to groups of neural precursors via Notch signaling. has expanded, whereas the size of the neural precursor and intermediate Here we show that the onychophoran nervous system is gen- neural precursor layers remains the same. Large arrowhead indicates a dif- erated by the massive irregular segregation of single neural ferentiated neuron; small arrowheads point to intermediate neural pre- fi cursors; arrowhead points to a segregated precursor. ms, mesoderm; np, precursor cells. Furthermore, we have identi ed homologs of the μ – μ euarthropod achaete-scute, Notch, and Delta genes in the ony- neuropile. (Scale bars: 25 minA C; 50 minD and E.) chophoran Euperipatoides kanangrensis and have analyzed their expression patterns in the embryonic ventral neuroectoderm. precursors in all stages of neurogenesis, indicating that the layer The data show that these genes are not regulated in a precise of segregated neural precursors is continuously replenished. spatiotemporal pattern as in euarthropods, and thus the selec- We observed scattered mitotic divisions in all layers of the de- tion of neural precursors is distinct in the onychophoran. veloping neuromeres except for the basal layers of differentiating Results neural cells (Fig. 2). Large mitotic cells are visible in the ventral neuroectoderm that are partially aligned in longitudinal and di- Morphology of Ventral Nerve Cord Formation. In the ovoviviparous agonal rows (Fig. 2 A and C). The segregated neural precursors onychophoran Euperipatoides kanangrensis the embryos develop seem to divide equally to generate two smaller intermediate in the uterus of the females and are presumed to be born ∼12 mo precursor cells (Fig. 2G, Inset). The intermediate neural pre- after fertilization (33). Thus, embryogenesis is considerably ex- tended as compared with that in euarthropods, in which em- cursors also proliferate, but are considerably smaller than the bryogenesis takes place within days or weeks. During early dividing neural precursors and neuroectodermal cells (Fig. 2B). We have analyzed the distribution of neural precursors that can embryogenesis in E. kanangrensis, the mouth and the anus fur- fi row form, and subsequently the germ band becomes distinct. The clearly be identi ed by their position basal to the neuroectoderm, their rounded shape, and the accumulation of F-actin in the cell two halves of the germ band become separated by presumptive – extraembryonic tissue, and bilateral segmental swellings with cortex (Fig. 2 D G and Fig. S1). At early stages, the neural pre- intersegmental groves become visible, which appear in an ante- cursors segregate from all areas of the ventral neuroectoderm rior-to-posterior sequence (26, 33, 34). The sequential formation (Fig. 2H). However, the formation of neural precursors becomes of segments correlates with an anterior-to-posterior gradient of restricted to the center of the developing hemineuromeres during – development, which facilitates the analysis of the sequence of further development (Fig. 2 G and I K), resulting in the gener- developmental processes. ation of ganglion anlagen, which can clearly be distinguished as The formation of neural precursors is first evident in the metameric units (Fig. 2 B and C). The number (ranging from ∼60 ventral neuroectoderm at late stage II [stages after Walker and to >100) and arrangement of neural precursors is different in Tait (33)], when the head and the first five trunk segments have each developing hemineuromere within the same embryo but also formed. Single cells delaminate from the neuroectoderm and between embryos of the same stage (Fig. 2 H–K). form a loose basal layer between the outer ectoderm and the inner mesoderm (Fig. 1A). Due to the segregation of additional Expression Pattern of the E. kanangrensis achaete-scute Homolog neural precursors, the basal layer increases to about two cells in EkASH. We have identified a single EkASH homolog in E. width but does not expand further during neurogenesis (Fig. 1 B kanangrensis (Fig. S2). EkASH is expressed before formation of and C). In the basal position, the neural precursors divide to neural precursors in newly formed segments (Fig. 3A). The ex- generate smaller intermediate neural precursors (Fig. 1B). The pression is initially homogenous in the developing neuromeres, latter form a second basal layer of approximately three to four but transcripts increasingly accumulate in the center of the neu- cells in width, and the innermost intermediate neural precursors romeres during further development (Fig. 3 A and B). A detailed differentiate into neural cells. Neurons extend their processes analysis of the EkASH expression pattern in the different layers of into the most basal lateral area of the developing hemi-neuro- the developing neuromeres revealed that the stronger expression meres to form the neuropile (Fig. 1D). During neurogenesis, the corresponds to an accumulation of transcripts in the segregated basal area of differentiating neurons expands, whereas the size of neural precursors, rather than an up-regulation of expression in the the neural precursor and intermediate neural precursor layers ventral neuroectoderm (Fig. 3 C–G). In the neuroectoderm, EkASH remains the same (Fig. 1E). We detected segregating neural is expressed at low levels throughout neurogenesis (Fig. 3 C–G),

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1008822108 Eriksson and Stollewerk Downloaded by guest on October 2, 2021 Fig. 3. (A–F) Expression pattern of EkASH. Light micrographs of flat prepa- rations (A–D) and transverse sections (E and F) of embryos stained with a DIG- labeled EkASH probe; anterior is toward the left in A–D, dorsal is toward the Fig. 2. (A–K) Proliferation and segregation pattern of neural precursors. top in E and F.(A and B) Ventral views show that EkASH expression is ho- Confocal micrographs of flat preparations of embryos at stage IV stained with mogeneous in the neuroectoderm. Transcripts are up-regulated in segre- phalloidin (red) and anti–phospho-histone 3 (blue) (A–G) and schematic gated neural precursors (arrows). Circles indicate the position of the limb drawings of neural precursors (H–K); anterior is toward the left in A–C and buds. Arrowheads in B indicate the area between the neuromeres which toward the top in D–K. (A) Mitotic cells (arrow) in the ventral neuroectoderm. shows low/absent levels of EkASH.(C and D) Sagittal views show that EkASH is (B and C) Formation of the ganglion anlage; the arrowheads indicate the expressed at low, homogenous levels in the neuroectoderm but is strongly segmental borders. Arrow in B points to proliferating intermediate neural expressed in the segregated neural precursors (arrows). Arrowhead indicates precursors; arrow in C points to a dividing neuroectodermal cell. (D–G) Op- the area between the developing neuromeres. (E and F) EkASH is strongly ex- EVOLUTION tical horizontal sections through the developing neuromere of the 2. walking pressed in the segregated neural precursors (arrow in E) and the intermediate leg segment of a stage IV embryo at different apico-basal levels (1–13 μm). precursors (arrowhead in F). l1 to l4, developing neuromeres of walking leg – Arrow in D indicates a dividing neuroectodermal cell; arrows in E–G point to segments 1 4; ml, ventral midline; ms,mesoderm;vne, ventral neuroectoderm. μ μ μ μ segregated neural precursors; arrowhead in G indicates a dividing neural (Scale bars: 100 minA, 100 minB,50 minC, and 50 minE and F.) precursor. Inset in G shows a higher magnification of dividing neural pre- cursors (arrowheads). (H–K) Pattern of segregated neural precursors in three consecutive hemisegments of the same embryo, representing consecutive Discussion developmental stages (stage IV/1–3) and the segregation pattern in an older Neurogenesis in the Onychophoran E. kanangrensis Does Not Reflect embryo (stage IV/late). Each schematic drawing represents the distribution of the Ancestral Pattern of Euarthropod Neurogenesis. Our data segregated neural precursors in a single hemineuromere and is based on the demonstrate that neurogenesis in onychophorans does largely analysis of stacks of 34–70 horizontal sections depending on the thickness of fl the neuromeres, which increases over time (Fig. S1). A, anterior; l1 to l4, de- not re ect the ancestral pattern of euarthropod neurogenesis. In veloping neuromeres of walking leg segments 1–4; P, posterior. (Scale bars: all four euarthropod groups, neural precursors are generated at 50 μminA and D–G;25μminB and C.) stereotyped positions despite the differences in neural precursor formation (NPGs versus neuroblasts). In each hemineuromere, a fixed set of ∼30 NPGs/neuroblasts segregates in a pattern that and expression becomes increasingly confined to the center of is similar in all euarthropod representatives that have been an- the developing hemineuromeres (Fig. 3C). Furthermore, there is alyzed (35, 36) (Fig. S5 B–D). The conservation strongly suggests a clear border of EkASH expression between the ventral neuro- that this pattern has been present in the last common ancestor ectoderm and the dorsal ectoderm from which the limb buds of euarthropods. develop (Fig. 3 A and B). After division of the segregated neural In contrast, in E. kanangrensis, a large number of neural pre- precursors, EkASH remains expressed in the intermediate neural cursors segregate from the ventral neuroectoderm. The number precursors (Fig. 3F), but expression cannot be detected in the and arrangement of neural precursors is different in each hemi- differentiating neural cells (Fig. S3). neuromere within the same embryo and also between embryos of the same stage. These data suggests that the neural precursors in Expression Pattern of the E. kanangrensis Notch and Delta Homologs. onychophorans segregate in a random, irregular pattern (Fig. We have identified single Delta and Notch homologs in E. S5A). If neural precursors were generated in stereotyped posi- kanangrensis that are most similar to the Notch and Delta tions, one would expect to see a repetitive pattern in contiguous homologs of two chelicerates, the cattle tick Boophilus microplus developmental stages, given the long phase of neurogenesis in and the spider Achaearanea tepidariorum, respectively. Similar to E. kanangrensis. EkASH, EkDelta is expressed before formation of neural pre- The regular arrangement of neural precursors in euarthropods is cursors; however, expression is clearly limited to the center of the reflected in the expression patterns of the proneural and neuro- developing neuromeres (Fig. 4A). EkDelta expression remains genic genes that are responsible for the controlled formation of the homogenous within these areas throughout neurogenesis (Fig. 4 neural precursors. In all euarthropods, proneural gene expression B and C). In contrast to EkASH, EkDelta is expressed strongly is up-regulated in proneural fields (clusters) that appear in in- not only in the neural precursors but also in the ventral neuro- variant positions in the ventral neuroectoderm. A restricted, fixed ectoderm and the mesoderm (Fig. 4 D and E and Fig. S4). number of neural precursors is generated in a regular sequence due EkNotch shows the same temporal and spatial expression pattern to the activity of Notch signaling. In contrast, the onychophoran as EkDelta; however, in contrast to EkDelta, transcripts can be achaete-scute homolog is neither expressed in proneural fields nor detected in the areas between the developing neuromeres in up-regulated in single neural precursors before their delamination; later stages (Fig. 5 A–C). Furthermore, EkNotch is expressed in rather, EkASH is expressed homogeneously in the whole neuro- differentiating neurons and the neuropile in the most basal area. ectoderm. This expression pattern suggests that neural precursor

Eriksson and Stollewerk PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 cells that subsequently segregate and strongly express the pro- neural gene. We conclude that the selection of neural precursors, as well as their arrangement and number, are conserved in all four euar- thropod groups, suggesting that this pattern has been present in the last common ancestor of euarthropods, although onycho- phorans do not share these neural characters.

Do the Neural Characters of Onychophorans Support the Myriochelata Hypothesis? Our morphological data on neurogenesis in E. kanangrensis are in line with recent publications on neurogenesis in other onychophoran species that describe the segregation of single neural precursors from the ventral neuroectoderm (24–26). In the basal position, the neural precursors divide to generate smaller intermediate precursors. We did not observe unequal – fl divisions of the neural precursors, which is in line with Mayer Fig. 4. (A E) Expression pattern of EkDelta. Light micrographs of at ’ preparations (A–E) and transverse sections (E) of embryos stained with and Whitington s results (25). This suggests that the neural pre- a DIG-labeled EkDelta probe; anterior is toward the left in A–D, dorsal is cursors do not divide in a stem cell-like manner; rather, each toward the top in E. (A–C) EkDelta is strongly expressed in the ventral neural precursor divides only once to generate two intermediate neuroectoderm (arrows). Expression is lower between the developing neu- precursors. This assumption is further supported by the fact that romeres (arrowheads). EkDelta is expressed in the sensory organ precursors. the neural precursor layer does not expand despite the continuous (D) Sagittal view of a stage II embryo shows that EkDelta is expressed in the segregation of a huge number of neural precursors. Similarly, the ventral neuroectoderm, segregated neural precursors, and mesoderm. intermediate neural precursor layer does not increase in size Arrowheads indicate area between segments. (E) At stage IV, EkDelta is during development, whereas the basal layer of differentiating expressed in the ventral neuroectoderm and segregated (arrows) and in- neural cells expands considerably. This again suggests that the termediate neural precursors (arrowheads) l1 to l6, developing neuromeres of walking leg segments 1–6; ml, ventral midline; ms, mesoderm; sop, sen- proliferation of intermediate neural precursors is limited. sory organ precursors; vne, ventral neuroectoderm. (Scale bars: A. 100 μm; Mayer and Whitington (25) suggest that the segregation of B, 50 μm; C, 100 μm; D, 50 μm; and E, 50 μm.) single neural precursors in the central nervous system and the generation of both neural and epidermal cells from the ventral neuroectoderm of onychophorans support a sister group re- formation in onychophorans is not regulated by spatiotemporal lationship of chelicerates and myriapods (Myriochelata). Cheli- cues as in euarthropods, which is in line with our morphological cerates and myriapods do not exhibit these neural characters but data that imply a continuous, irregular segregation of neural pre- share the segregation of NPGs and the exclusive formation of cursors in onychophorans. Furthermore, EkASH is strongly ex- neural cells from the central area of the ventral neuroectoderm pressed in the delaminated neural precursors, whereas the (10, 11). However, we do not agree with Mayer and Whitington for proneural genes of euarthropods are down-regulated after the the following reasons. First, the nature of the neural precursor segregation of NPGs/neuroblasts. These expression data suggest cells is different in onychophorans and Tetraconata (insects/ different functions of the onychophoran proneural genes as com- crustaceans). In insects and higher crustaceans (malacostracans), pared with euarthropods. We assume that the coexpression of the individual neuroblasts are specified in the neuroectoderm, which proneural and neurogenic genes in the ventral neuroectoderm divide several times in a stem cell-like mode to generate ganglion results in low levels of EkASH transcripts due to transcriptional mother cells. In insects, single neuroblasts delaminate from the repression by the effectors of the Notch signaling pathway. Subtle, ventral neuroectoderm before they start producing ganglion random differences in the expression levels of EkASH and/or mother cells, whereas malacostracan neuroblasts do not de- EkDelta might lead to a reduction in Notch signaling in individual laminate but proliferate in the outer neuroectoderm. In contrast, onychophoran neural precursors cannot be considered as stem cells, as they seem to divide only once to produce two intermediate precursors. Thus, they show only superficial similarity to insect neuroblasts, because they delaminate individually, but do not share any similarities with crustacean neuroblasts. Furthermore, the formation of neural precursors is different in onychophorans and Tetraconata. Insect and crustacean (malacostracan) neuro- blasts arise from a limited number of proneural clusters that are set up in stereotyped positions, whereas neural precursors are formed in irregular, adjacent positions in onychophorans. Second, the formation of neural precursor groups in many areas of Drosophila neurogenesis strongly suggests that NPGs are part of the ancestral pattern of euarthropod neurogenesis and therefore cannot be used to resolve euarthropod relationships. The pars intercerebralis and pars lateralis, for example, are part Fig. 5. (A–C) Expression pattern of EkNotch. Light micrographs of flat of the central neuroendocrine system in the Drosophila brain and preparations (A and B) and transverse section (C) of embryos stained with DIG- derive from NPGs that invaginate and become attached to the labeled EkNotch probe; anterior is toward the left in A and C; dorsal is toward brain primordium (37). Additional examples are the stomato- the top in B. (A and C) EkNotch is expressed homogenously in the ventral gastric nervous system, which is generated by three large NPGs, neuroectoderm (arrows). (B) In addition, EkNotch is expressed in the segre- the bilateral invaginations of the optic primordium and the gated (arrow) and intermediate neural precursors (arrowhead), the neuropile generation of large precursor groups in the developing poly- and the most basal differentiating neurons (small arrowheads), and the me- – soderm (small arrows). l1, l9 to l11, developing neuromeres of walking leg clonal sensory organs of Drosophila (38 40). segments 9–11; ml, ventral midline; ms, mesoderm; vne, ventral neuro- Finally, based on the following data, we suggest that the ex- ectoderm. (Scale bars: A and C, 100 μm; B, 50 μm.) clusive generation of neural cells from the central area of the

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1008822108 Eriksson and Stollewerk Downloaded by guest on October 2, 2021 ventral neuroectoderm of chelicerates and myriapods represents If we assume that the presented evolutionary sequence is cor- an ancestral character of arthropod neurogenesis. First, in ony- rect, we would have to conclude that neuroblasts have gradually chophorans, this mode of neurogenesis can be observed in the evolved from NPGs. There is indeed evidence from intermediate ventral protocerebral primordium. In this area, the whole neu- states that supports this theory. In the centipede Strigamia mar- roectoderm becomes internalized and forms vesicles that have itima, all cells of a NPG are attached to a single cell of the group been termed hypocerebral organs. Neural precursors continue to rather than to the apical surface as in chelicerates and millipedes segregate from the hypocerebral organs after their invagination (14). This cell expresses higher levels of Delta transcripts as com- (24). Second, in Drosophila, the embryonic procephalic neuro- pared with the remaining cells of the group. Thus, Delta/Notch ectoderm that generates the larval brain neuromeres generates signaling seems to generate single cells with distinct properties almost exclusively neural cells. The epidermis extends over the within the NPGs in Strigamia, although the whole group eventually brain primordium during head involution (41). Furthermore, invaginates and gives rise to neural cells. Furthermore, in the basal fundamental differences in the formation of epidermal cells in insect Schistocerca americana, groups of four to eight cells are the ventral neuroectoderm of insects and malacostracans suggest selected in the ventral neuroectoderm (12). One cell of the group, that the capacity to generate epidermal and neural cells has evolved independently in insects and crustaceans. In insects, the the neuroblast, delaminates, whereas the remaining cells of the cells that remain in the neuroectodermal layer after segregation group differentiate into a cap cell and sheath cells that surround of the neuroblasts develop into epidermis. In contrast, in mala- the neuroblast and its lineage. costracans, neuroblasts remain in the neuroectoderm and can generate both neural and epidermal cells (13, 28). Conclusions. Our data on onychophoran neural precursor formation contradict neither the Mandibulata nor the Myriochelata hypoth- Hypothesis on the Evolution of Neurogenesis in Arthropods. Based esis. However, we suggest that the invagination of NPGs and the on the data presented here, we suggest the following evolution- exclusive generation of neural cells in the ventral neuroectoderm of ary scenario of arthropod neurogenesis. In the last common chelicerates and myriapods represent plesiomorphic characters that ancestor of arthropods, the nervous system was generated by cannot be used to support the Myriochelata hypothesis. Further- a massive irregular segregation of individual neural precursor more, the deployment of onychophoran characters for outgroup cells (Fig. S6). Proneural genes might have been expressed ho- comparison has to be considered carefully. Crown-group euar- mogeneously in the whole neurogenic area to confer neural thropods and probable stem-group onychophorans already existed potential to all cells. We speculate that the whole neurogenic in the early Cambrian period (46–48). Hence, the onychophoran EVOLUTION area exclusively generated neural precursors, similar to the case lineage must have had a long evolutionary history diverged from the in chelicerates, myriapods, and the brain primordia of Drosophila euarthropod stem lineage. Therefore, extant onychophorans might and onychophorans. not show the ancestral state of neurogenesis without modifications. Cell proliferation might have occurred mainly in the neuro- Indeed, our data show that the development of the nervous system ectoderm. Individual neural precursors became postmitotic, seg- in onychophorans largely does not reflect the ancestral pattern of regated from the ventral neurectoderm and directly differentiated euarthropod neurogenesis; rather, it seems to show a mixture of into neural cells. This hypothesis is supported by the nature of the derived characters and ancestral characters that have been present neural precursors in chelicerates and myriapods. In both euar- in the last common ancestor of arthropods but have been modified thropod groups, the NPGs are postmitotic and directly differen- in the euarthropod lineage. tiate into neural cells after their segregation. This ancestral state has been retained in the stomatogastric nervous system of Dro- Materials and Methods sophila melanogaster, which is generated by neural precursors that Collection, Animal Husbandry, and in Situ Hybridization. Collection and cul- invaginate as large groups and directly differentiate into neurons turing of female Euperipatoides kanangrensis Reid, 1996 was done as de- (38). The hypothetical ancestral state is further supported by re- scribed previously (48). In situ hybridization was carried out as described by cent data on neurogenesis in an annelid (42). Annelids belong to Eriksson et al. (49). the Lophotrochozans, which are a sister group to the Ecdysozoa [e.g., arthropods, nematodes (43)]. In the polychaete Capitella sp. Isolation and Sequencing of E. kanangrensis Genes. We isolated fragments of I, one to several adjacent neural precursors delaminate from the genes from embryonic cDNA libraries, cDNA, and genomic DNA. Degenerate procephalic neuroectoderm. The neural precursors do not divide primers for EkDelta were as described in Stollewerk (32) and for EkASH as de- after their delamination; rather, they directly differentiate into scribed in Stollewerk et al. (10). EkNotch was amplified by using specific primers neural cells. Thus, subsequent stages of neurogenesis must have constructed from a Notch homolog sequence in another species of Onycho- diverged in the lineage leading to the onychophorans, such as phora, Euperipatoides rowelli; this sequence was kindly supplied by Georg mitotic divisions of the segregated neural precursor cells, the Mayer (Institute of Biology II: Animal Evolution & Development, University of Leipzig, Leipzig, Germany). These fragments were extended by nested PCR evolution of intermediate neural precursors, and the generation fi fi of epidermal and neural cells from the ventral neuroectoderm. using an embryonic cDNA library with gene speci c primers and vector speci c primers as well as RACE using the 5′ 3′ RACE kit from Roche. The sequences have In the lineage leading to the euarthropods, molecular changes been deposited in the GenBank database with the following accession num- in the proneural genes occurred, facilitating the spatiotemporal bers: EkASH, GU954550; EkDelta, GU954551; and EkNotch, GU954552. regulation of these genes, and allowed for the up-regulation of fi transcripts in proneural elds at stereotyped positions (Fig. S6). Histological Staining. For the detection of F-actin, we used Alexa phalloidin 488 The segregation of neural precursors within these fields was (Invitrogen); 2 U dissolved in 10 μL methanol was evaporated to remove the synchronized. Continuous refinement of proneural gene regula- methanol and then resuspended in 200 μL PBS and added to the embryos. tion, possibly by evolutionary changes in the regulatory regions, After 1 h, the embryos were washed in PBS and used for imaging. To detect duplication and divergence of proneural genes (44, 45), and the mitotic cells, we used the polyclonal rabbit anti-alpha phospo histone 3 an- incorporation of Notch signaling resulted first in the invagination tibody (Sigma) diluted 1:200 times in PBS with 0.5% BSA and 0.1% Triton ×100. of small groups of neural precursors at fixed positions, as seen in Antibody incubation was done as described in Eriksson and Budd (50). chelicerates and myriapods, and subsequently in singling out of individual neuroblasts as seen in insects and crustaceans (Fig. S6). ACKNOWLEDGMENTS. We thank the members of the A.S. laboratory for continuous stimulating discussions and Petra Ungerer for critical comments The capacity to generate both neural and epidermal cells from on the manuscript. We are grateful to Edina Balczo for excellent technical the ventral neuroectoderm was probably achieved independently support. We thank Noel Tait for help with collecting animals. This work was in insects and crustaceans. supported by the Biotechnology and Biological Sciences Research Council.

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