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 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. 22576–22581 | PNAS | December 28, 2010 | vol. 107 | no. 52 www.pnas.org/cgi/doi/10.1073/pnas.1008822108 Downloaded by guest on September 28, 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.