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Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 833

Evolution and Development of the

Onychophoran Head and

BY JOAKIM ERIKSSON

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400 ##%86 & < 405 3#677867$#&62  '  ''' 6&&2= > '?? ),)? @ A '  ''' 6&&2=B This thesis is based on the following papers.

I. Eriksson BJ, Budd GE. 2001. Onychophoran cephalic and their bearing on our understanding of head and stem-group evolution of Arthropoda. Structure & Development 29:197- 209.

II. Eriksson BJ, Tait NN, Budd GE (2003) Head Development in the Onychophoran Euperipatoides kanangrensis With Particular Reference to the Central Nervous System. Journal of Morphology 255:1-23

III. Eriksson BJ, Tait NN, Budd GE An Ultrastructural Investigation of the Hypocerebral Organ of the Adult Euperipatoides kanangrensis (, Peripatopsidae). Manuscript.

IV. Eriksson BJ, Larson E, Tait NN, Budd GE Expression of engrailed in the developing and appendages of the onychophoran Euperipatoides kanangrensis (Reid). Manuscript.

Reprints of papers I and II were made with the permission from the publishers. First author is the main contributor to the papers.

Contents Introduction...... 7 Onychophorans in general...... 7 Phylogeny...... 10 Classification and distribution of the Onychophora ...... 10 Arthropod phylogeny and the versus the Articulata concept ...... 11 The Acron...... 12 Model organisms...... 13 Arthropod head segmentation ...... 13 Development of the nervous system ...... 15 The record...... 17 characters of onychophorans...... 18 Evolution and development...... 20 Introduction to the present investigation...... 21 Methodology ...... 23 Detection and description of cephalic nerves ...... 23 Embryological investigations ...... 24 Results and discussion ...... 25 Innervation of the head (paper I)...... 25 Development of the nervous system (paper II) ...... 25 The hypocerebral organ (paper III) ...... 26 Expression of the gene engrailed during development (paper IV)...... 26 Conclusions...... 27 Acknowledgements...... 29 References...... 31

Abbreviations

CLSM Confocal Laser Scanning Microscope DIG Digoxygenin HRP Horse-Radish Peroxidase PCR Polymerase Chain Reaction SEM Scanning Electron Microscope TEM Transmission Electron Microscope

Introduction

Onychophorans in general The following comments are intended as a short introduction to the general morphology of the Onychophora; for a more detailed description refer to Storch and Ruhberg (1993). Onychophorans, commonly referred to as peripatus after the first described (Guilding 1826), or velvet because of the texture of their skin, consist of approximately 150 located on land-masses derived from the fragmentation of the supercontinent Gondwanaland. This biodiversity includes a large number of species recently described from Australia (Reid, 1996). Onychophorans are , 5 mm to 15 cm in length, with short lobopodial limbs segmentally arranged down the length of the body (Fig. 1).

Fig. 1 Cephalofovea clandestina, an onychophoran from Australia. Photo by Mats Block.

The head carries a pair of large and prominent antennae situated anteriorly and dorsally. At the base of each antenna is a simple ocellal eye (Fig. 2). The mouth is located ventrally and is surrounded by lips equipped with sensory spines (Fig. 3). A pair of sclerotized jaws is situated inside the oral cavity together with a muscular tongue that is an expanded part of the

7 dorsal (Fig. 3). At the posterior part of the head there is a pair of slime papillae that eject slime for defensive as well as offensive (i.e. prey capture) purposes (Fig. 2).

Fig. 2 A, Light micrograph of an adult onychophoran of the species Euperipatoides kanangrensis showing the anterior end with associated structures and appendages; A, antenna; M, mouth; L1, leg 1; L2, leg 2; Sp, slime papilla; the arrowhead point towards the eye. Scale bar = 1 mm. Dorsal is up and ventral down. B, SEM micrograph of the eye (E) of Euperipatoides kanangrensis surrounded by dermal papillae (Dp) with and without sensory bristles (arrow head). Scale bar = 50 µm.

The integument is thin, flexible and covered by a chitinous cuticle that is moulted regularly. The surface is covered with dermal papillae, the larger ones carrying a sensory bristle (Fig. 2). Onychophoran integument is water repellent, although it gives little protection against desiccation. Hence, onychophorans live in moist terrestrial microhabitats such as under rocks, in leaf litter or inside rotting logs. The colour of onychophorans is variable, often bright and delicately ornamented. The body contains no hard parts, except for the jaws and claws, and muscles work against a hydrostatic skeleton. The muscles of the trunk, which are located just inside the connective tissue of the integument, are oriented as circular, oblique and longitudinal bands. The onychophoran trunk also has internal transverse muscles that divide the body cavity into a median and two lateral compartments. The central nervous system is composed of a large brain, circumesophageal connectives, and paired ventro-lateral cords that run homogeneously without any concentrations into ganglia in the body (Fig. 4). Evenly distributed commissures, approximately 10 in each “segment”, connect the individual nerve cords. is conducted by metanephridia that have openings at the base of each leg, and opens

8 internally into coelomic sacs called end sacs. The sexes are separate and fertilisation is internal with viviparous, ovoviviparous or oviparous modes of development. are transferred as spermatophores which, in some species, are deposited directly on the surface of the female body. Blood cells from the female digest the integument so that sperm enter the body cavity and migrate towards the for fertilisation of the oocytes or storage in seminal receptacles. In other species spermatophores are transferred directly to the female gonopore by special structures on the head of males (Tait and Norman, 2001).

Fig. 3 SEM micrograph of the head of E. kanangrensis as seen from the ventral side with jaws (lower arrowhead), tongue (upper arrowhead), oral lips (O), slime papilla (Sp) and the antenna (A). Scale bar = 500 µm.

Onychophorans have an open circulatory system with a dorsal heart. The blood contains two categories of cells, haemocytes (amoebocytes) and nephrocytes. No respiratory pigments have been reported; cells and tissues are supplied with oxygen from tracheal tubules. The spiracles of the tracheae occur over the general body surface and cannot be closed, a contributing factor that together with loss of moisture through the epidermis renders onychophorans prone to desiccation. Onychophorans live in hidden habitats, avoid light and show nocturnal behaviour. Their prey consists mainly of small , which they immobilize with their sticky slime. Prey is torn open by the sickle-shaped jaws and digestive saliva is injected into its body.

9 The digestive system consists of a straight gut that is divided into fore- gut, mid-gut, and hind-gut. Fore-gut and hind-gut are lined with cuticle that is shed periodically. There are no digestive diverticula present in the digestive system and, to a great extent is believed be accomplished outside of the body by the saliva that is injected into the prey.

Fig. 4 A light micrograph of a dissected-out E. kanangrensis brain (B) with part of the nerve-cord (Nc) attached. An, antennal nerve; C, commissure; Coc, circum- oesophageal connective; E, eye. Scale bar = 0.5 mm

Phylogeny

Classification and distribution of the Onychophora Living representatives of the Onychophora are divided into two families, the Peripatidae (Evans, 1901) and Peripatopsidae (Bouvier, 1907). Members of Peripatidae occur in tropical regions of West Africa, southeast

10 Asia, Central and South America. Peripatopsidae occupy temperate regions of South Africa, Australasia and Chile. The two families probably diverged before the break-up of Gondwanaland since both families are present in South America and South Africa (Ghiselin, 1985).

Arthropod phylogeny and the Ecdysozoa versus the Articulata concept Onychophora, together with the Tardigrada and Euarthropoda, comprise the Arthropoda, a grouping that is well supported (Nielsen, 2001). A few of the synapomorphies uniting the Arthropoda can be mentioned here: cuticle with α-, food manipulated with modified limbs, segmentally arranged appendages, mixocoel with a dorsal heart and ecdysis. do not have a heart, but this can be regarded as a secondary loss because of their small size; and the chemical composition of their cuticle is not known. Cladistic analyses based on molecular characters have also corroborated the Arthropoda (Ballard et al., 1992; Boore et al., 1995; Aguinaldo et al., 1997; Edgecombe et al., 2000). Furthermore, some of the molecular analyses have suggested a broader grouping termed the Ecdysozoa (see e.g. Aguinaldo et al., 1997 and Schmidt-Rhaesa et al., 1998). The Ecdysozoa unites Arthropoda, Tardigrada, Onychophora, Nematoda, , , and into one clade. The Ecdysozoa hypothesis is not supported by many morphological characters, although some have emerged e.g. ecdysis and a tri-radial pharynx (see e.g. Schmidt- Rhaesa et al., 1998; Manuel et al., 2000). However, since several molecular investigations conclude that the Ecdysozoa is valid, it is obvious that the morphological characters must be re-analysed with the Ecdysozoa concept in mind. A phylogeny based on molecular characters without morphological characters to support them is not well-founded, and vice versa. The opposing and classical view argues for a grouping of the Annelida with Arthropoda with a number of uniting characters; posterior ectodermal and mesodermal growth zone (see Fig. 5), segmentally developing coelomic sacs, and segmental paired ganglia are examples of important developmental characters (Scholtz, 1997, 2002, 2003). Onychophora has for a long time been regarded as a link between the two groups, with characters of both; α- chitin, ecdysis, and trachea are examples of arthropod characters; whereas body-wall muscles acting against a fluid filled body cavity (hydrostatic skeleton) and the ciliated funnels of the metanephridia are similar to characters (Nielsen, 2001).

11 The Acron Another character of some importance when discussing the Ecdysozoa versus the Articulata concepts is the so-called acron. In , especially the , the acron is a prominent structure at the anterior end of the head. This is especially obvious in the trochophora of polychaetes (Fig. 5). The newly hatched trochophora larva is a small, round, slightly elongated organism with a mouth located in the middle of the body and with the at the posterior end (Fig. 5).

Fig. 5 A generalized trochophora larva showing the position of the mouth (M) and the anus (A) in relation to the anteriorly placed acron (Ac). Gz, growth zone.

The area anterior of the mouth is referred to as the acron. The acron contains, among other things, the brain of the trochophora larva, the so- called apical organ. Later during the ontogeny, additional segments with nervous system add to the growing larva from a posterior pre-anal growth zone (Fig. 5) until a complete is formed, this growth zone bearing similarities to that of arthropods (Scholtz, 1997). The brain of annelids is thus largely derived from the anteriorly-placed unsegmental acron (Hanström, 1935). Because the traditional view has been that annelids and arthropods together form a group, the Articulata, it has been envisaged that the acron constitutes the anterior part of the arthropod head too. Hence, varying parts of the arthropod head together with neuromeres and appendages have been interpreted as unsegmental, even though they apparently develop in a segmental fashion (e.g. the antenna) (see e.g. Hanström, 1935; Butt, 1960; Snodgrass, 1960). The nature of the most anterior region of the arthropod head (ocular-protocerebral) is far from clear and it is not clear if it is an acron or a true segment (Scholtz, 2003). If the Ecdysozoa concept is accepted, a different picture may emerge. At least one

12 clade of the Ecdysozoa, i.e. the , has a terminal mouth, and hence, an unsegmental anterior acron cannot exist in the Ecdysozoa model, unless the terminal mouth is a synapomorphy of the Cycloneuralia. It has also been suggested that the basal position of the mouth in arthropods is terminal, and that the nerves that support the stomatogastric system have been transferred from an anterior and terminal position to a more posterior neuromere (Dewel et al., 1999). It is generally regarded that the ground-state of the position of the arthropod mouth is ventral, with the terminal mouth in tardigrades being secondarily derived. This assumption, however, is based on the Articulata concept, since the most parsimonous explanation of the basal mouth position in the Articulata is ventral. Data from a more basal arthropod, such as Onychophora, will perhaps give indications on the basal mouth position of the arthropods.

Model organisms Most of modern evolutionary developmental research is concentrated on so- called model organisms. This is, of course, the only feasible option when continuous and abundant supplies of specimens are needed; for example when complicated genetic mechanisms or manipulative approaches to morphological research, such as transplantation experiments, are conducted. Research done on organisms such as the Drosophila melanogaster, the elegans, the amphibian Ambystoma mexicanum, or the mammal Mus musculus have contributed tremendously to present knowledge in this field of science. However, it is essential for a deeper understanding of the evolution of the present day phyla to investigate and compare a broader spectrum of organisms. Comparisons based on a broad sample of organisms will more likely lead to a correct assessment of a basal character-state.

Arthropod head segmentation In arthropods the body is divided into functional units called tagmata. Originally, each of these tagmata was composed of individual segments with a pair of appendages; these segments were merged during evolution into functional units (Cisne, 1974; Scholtz, 1997; Wills et al., 1997; Budd, 2000). Today some tagmata, e.g. head or opisthosoma, of certain arthropod groups are impossible to separate into actual segments in the adult . This is probably the reason why arthropod head segmentation has been such a debated topic during the last century (see e.g. Rempel, 1975).

13 With the discovery of new techniques to visualize morphology and gene expression, such as immunohistochemistry and in situ hybridizations, came a renaissance of interest in the problem. The discovery of important developmental genes such as the so-called Hox-genes and the segment polarity genes has also fuelled the debate. The Hox-genes are expressed in a co-linear fashion and the anterior-posterior sequence by which they are expressed in the embryo is conserved among bilaterians (see e.g. Carroll, 1995). The anterior Hox-genes, grouped together as the antennapedia complex, have anterior expression borders that seem to coincide with the boundaries of the original head segments of the last common ancestor of and chelicerates (Damen et al., 1998; Telford and Thomas, 1998). The expression of the segment polarity genes engrailed and wingless has led researchers to make conclusions about the number of head segments in for instance Drosophila (Schmidt-Ott and Technau, 1992) and in (Scholtz, 1995, 1997). However, the very complicated morphology of the arthropod head makes it difficult to be certain about the number and topology of the head segments. Therefore it seems more plausible to identify and analyse possible arthropod stem-group members as well as basal members of the Arthropoda, i.e. Onychophora, in order to explain the early steps of tagmatisation. A structure that has been particularly difficult to put into context, and therefore is of special interest, is the anterior structure of the euarthropod head referred to as the labrum. The labrum has been proposed to be an appendage of the ocular segment (Rempel, 1975), an appendage of the tritocerebral segment (Haas et al., 2001a, 2001b) or a very anterior, unsegmental outgrowth of euarthropods (Scholtz, 2001). The onychophorans, being basal arthropods, might be able to provide some insight into this problem, but the nature of the onychophoran head has remained a puzzle. The few investigations of the onychophoran anterior part that do exist (e.g. Hanström, 1935; Henry, 1948) are contradictory and they are obviously influenced by the Articulata concept, which was prevalent at that time. Hanström (1935) and Henry (1948) concluded that the antennae in onychophorans, being innervated from the most anterior part of the brain, are homologous to the anterior palps of annelids. The basis for this assumption is that the anterior part of the arthropod brain constitutes the archicerebrum, which is a derivative from the acron of annelids according to the Articulata concept. Hanström (1935) believed that the homologue to the brain of the annelids, that innervates the palps, is to be found in anterior part of the arthropod brain. This is by no means an unreasonable interpretation, but with the present day knowledge about the Middle lobopods, which are likely to be members of the arthropod stem-group (Budd, 1996, 2002), it is difficult to maintain the existence of an anterior unsegmental

14 acron, since none of these Cambrian lobopods possessed such a structure. It is therefore more likely that the antennae of onychophorans are homologous to some segmental appendage of euarthropods, and an investigation of the anterior part of the onychophoran head is needed in order to identify such a homologue.

Development of the nervous system The nervous system is derived from the ectoderm in all animals. The ectoderm, however, also gives rise to additional tissues such as integument, part of the eyes etc. Therefore, it is obvious that those cells that will give rise to nervous tissue, as distinct from other tissues, must be committed to a nerve-tissue. The part of the ectoderm that gives rise to nervous tissue is called neuroectoderm (Fig. 6).

Fig. 6 A schematic drawing of the neuroectoderm (N) in an early onychophoran embryo just after formation of mesodermal cavities (C). A cross section through a coelomic cavity with the overlying neuroectoderm that is just slightly thicker than the neighbouring extraembryonic ectoderm. Below the mesoderm surrounding the coelomic cavity is the yolk (Y) with intermingled trophoblasts.

In this neuroectoderm, a certain subset of cells continues their development as neuronal precursors. In insects and these cells have been demonstrated to be selected by a process known as lateral inhibition; cells selected for a neuronal destiny inhibit their neighbours from following a neurogenic path by secreted molecular cues (Lewis, 1996). Once committed to their neuronal destiny, these precursors will differentiate to become neuroblasts in insects (Anderson, 1973; Campos-Ortega, 1997) and crustaceans (Scholtz, 1992). Neuroblasts are cells that by unequal give rise to smaller ganglion mother cells; the ganglion mother cells in turn developing into either neurones or glial cells (Scholtz, 1992). During this

15 differentiation to neurones the cells concomitantly migrate from the superficial layers to deeper areas. The neuroectoderm in onychophorans develops from ectoderm situated outside of the mesodermal somites. The mesoderm is formed from a posterior growth zone, and then migrates anteriorly as sheets of cells encircling a cavity, the (Anderson, 1973). However, there is also an example of an onychophoran with simultaneous formation of the segments, a so-called long germ band (Walker, 1995). The ectoderm outside of the somite starts to proliferate when that somite has reached its final position (Anderson, 1973). Since the mesodermal sacs or somites separate after their formation in the posterior growth zone, and migrate forward on each side of the embryo, a band of proliferative cells will appear on each side (Fig. 7).

Fig. 7 A schematic drawing of a germ band stage onychophoran embryo with two segments formed, the antennal segment (A) and the jaw segment (J). The posterior part of the mesoderm is still forming a continuous germ band (GB) that is proliferating from the posterior growth zone near the anus (An).

These bands are the germ bands, and the germ band stage is sometimes claimed to be the so-called phylotypic stage of the Arthropoda (Sander, 1983, but see e.g. Scholtz (1997) for a criticism of the concept of a phylotypic stage). These germ-bands will give rise to the majority of the animal tissue. As the neurones differentiate they start sending out axons in order to connect to other part of the nervous system and commissures and connectives are formed as well as synaptic areas of nerve fibres. These areas, devoid of cell bodies (perikarya), are the so-called neuropiles. It has been

16 shown in euarthropods that when axons grow, they follow special paths guided by secreted molecules. Several of such guiding cues have been described and characterized, and one of the genes tat codes for such a potential axon-guidance cue, engrailed (Marie et al., 2002), has been investigated in the present study.

The fossil record Arthropods are unusual in that they have a remarkable fossil record compared to other groups of animals. The first arthropods are found in Lower Cambrian rocks, and they are already recognized as stem-groups members of the different classes of present day arthropods (Budd and Jensen, 2000). The Arthropoda exclusive of the Euarthropoda, on the other hand, has a more limited fossil record because of small size or lack of hard parts. Tardigrades are first described from the Middle Cambrian (Müller et al., 1995), and there is a finding in amber (Cooper, 1964). The Middle Cambrian specimens have, however, three pairs of leg as opposed to four pairs in recent tardigrades; this fact makes it more correct to place them in a stem-group rather as crown-group tardigrades. The Onychophora has a very sparse fossil record indeed; one possible example, which lacks the head with characteristic onychophoran synapomorphies, from the Late (Thompson and Jones, 1980) and also a poorly preserved specimen preserved in amber (Poinar, 1996). There are, however, several fossil representatives of probable stem-group members of the Arthropoda described from the Cambrian, some of which show an onychophoran grade of body organisation (Ramsköld and Hou, 1991; Chen and Zhou, 1997; Budd, 1996, 1999; Budd and Peel, 1998). Budd (2002) has presented a series of evolutionary events, based on fossil stem- group panarthropods and arthropods, which may explain how a complicated head as seen in the recent arthropods has been evolved from an animal such as Aysheaia with a head equipped with a terminal mouth and one pair of modified feeding appendages. Some aspects of such a hypothesis can be tested by investigating an organism that retains some of the basal characters once possessed by the members of the arthropod stem-lineage, and onychophorans are a good candidate for such a model.

17 Basal characters of onychophorans The Onychophora is often regarded in the literature as including forms from Cambrian strata, such as Aysheaia and . However, there are important characters that differ between these organisms, apart from five hundred million years of evolution. Characters unique to the recent Onychophora (derived characters or synapomorphies) are for example; strictly terrestrial (the Cambrian Onychophora-like organisms were all marine), the possession of jaws, and appendages modified to slime ejectors. Therefore, it is probable that a more correct image of evolutionary events emerges if the Cambrian Onychophora-like organisms are placed in a stem- lineage leading to present day onychophorans and arthropods (Budd and Jensen, 2000; and Fig. 8 herein). If, however, we are going to use the Onychophora as proxy for arthropod evolution, we need to identify basal characters, present in the last common ancestor of arthropods and onychophorans.

18

Fig. 8 Tree showing possible relationships among fossil and recent animals. Based on Schmidt-Rhaesa et al. (1998), Budd (1996, 1999), Budd and Jensen (2000) and Eriksson et al. (2003).

It is likely that the acquisition of a more complex body with specialised tagmata, such as seen among recent arthropods, was accompanied by the evolution of the nervous system to support such complex apparatuses. The nervous system of the Onychophora may prove to be the closest we can get as a glimpse to how the basal nervous system might have looked like in an early arthropod ancestor, since there is no sign of any nervous system left in the of early onychophoran-like animals. Therefore, it is necessary to

19 separate the changes that have occurred separately in onychophorans during their evolution from the last common ancestor of arthropods and onychophorans. This last common ancestor might very well have been very similar to the Cambrian Aysheaia. Aysheaia had a trunk that in the fossils look strikingly similar to an onychophoran trunk; a series of short stout, unsegmented legs on a homonomous body. The differences lie mainly in the head; Aysheaia had a terminal mouth, only one pair of modified appendages (possibly for a sensory function), and there is no sign of any eye (Whittington, 1978). Even though present day arthropods are all very complicated animals, it is perhaps possible to detect basal characters by comparing certain morphological aspects of different arthropod groups with their phylogenetic position. For example, efforts in this line have suggested that chelicerates, basal within the Arthropoda (Edgecombe et al., 2000), have a less derived mechanism by which neuronal precursor-cells are selected during embryogenesis (Stollewerk et al., 2001). Thus, possible basal onychophoran characters may include; the homonomous latero- without any ganglionic concentration supporting a series of unsegmented limbs, a head-tagma comprising three incorporated appendage bearing segments of which the affinity to anterior appendage bearing segments in arthropods is discussed in this thesis, and the development of the neuroectoderm, which is also discussed below.

Evolution and development With modern methods in molecular research has come an upsurge of new and old ideas in the field of evolutionary developmental or evolution of body-plans. Among such methods is, for instance, the use of antibodies to detect specific tissues on the basis of their protein content, e.g. acetylated α- tubulin in nerve fibres, or the detection of gene products that act as transcription factors, e.g. Hox-genes. The so-called Hox-gene group is perhaps one of the most important groups of genes in this respect. Hox-genes have been detected in all bilaterian groups examined. The systematic research done on this gene-cluster, in concert with concomitant analyses and re-evaluation of data obtained from morphological studies on recent and fossil material, has perhaps added most to the present day understanding of the evolution of body-plans (Hughes and Kaufman, 2002). Another gene that has gained much attention is the segment polarity gene engrailed. The mutated phenotype that gave name to the gene was first described in the fly Drosophila melanogaster in 1926 by Eker (1929). The name engrailed comes from the French word engrailé, literally translated as “dented by hail”, and refers to the notch in the scutellum of the mutant (Gibert, 2002). Engrailed was found to be expressed in the posterior compartment of

20 developing segments of Drosophila (Garcia-Bellido and Santamaria, 1972; Lawrence and Morata, 1976; Nüsslein-Volhard and Wieschaus, 1980), in crustaceans (Manzanares et al., 1993; Scholtz et al., 1993; Gibert et al., 2000), and in chelicerates (Damen et al., 1998).Workers have used engrailed as a molecular segmental marker because of the fact that it defines the posterior part of each segment, and have drawn conclusions about, for instance, the number of head segments in the insect (Schmidt-Ott and Technau, 1992) and (Scholtz, 1995, 1997) head. Later investigators have greatly added knowledge about the expression and function of engrailed; as involved in axon pathway- and trajectory choice (Marie et al., 2002), establishing neuroblast identity (MacDonald and Doe, 1997), patterning of the mouse limb-bud (Loomis et al., 1996), in the shell margins of molluscs (Jacobs et al., 2000), and even the possibility of engrailed acting in an paracrine way in the rat (Joliot et al., 1997, 1998). It has been suggested that the role of engrailed in neurogenesis dates back before the bilaterians diverged (Patel et al., 1989). The expression of engrailed has been examined in the onychophoran Acanthokara kaputensis with the help of polyclonal antibodies (Wedeen et al., 1997). The expression was found to be restricted to the posterior mesodermal part of the segments, and no clear staining of engrailed positive cells was found in the head or in the developing nervous system. The lack of staining in the developing nervous system would argue against engrailed having a basal role in neurogenesis, and the lack of expression in the anteriormost head segment is difficult to explain since engrailed is expressed in the eye-protocerebral region in insects (Scmidt-Ott et al., 1994), crustaceans (Scholtz, 1995) and myriapods (Hughes and Kaufman, 2002).

Introduction to the present investigation The aim of this investigation was to investigate certain aspects of the morphology of adult onychophorans as well as their embryology in order to answer some questions concerning Arthropod evolution:

1. The nature of the onychophoran antenna with regards to its relation to cephalic appendages within the Arthropoda: It was regarded as important to investigate the adult innervation of the antenna as well as its embryonic origin in order to assess a possible arthropod homologue. It is probable that the last common ancestor of arthropods and onychophorans possessed legs and it is also probable that these appendages were modified during evolution to take up functions as feeding and sensory appendages, rather than that these feeding/sensory appendages were acquired de novo. It is therefore reasonable to assume that segments with attached appendages

21 are serially homologous within the Arthropoda. However, although there is no example of de novo acquisition of segmental appendages in the head region, the reverse has surely happened. For instance, the deutocerebral segment in insects lacks an adult appendage, yet we can be certain that an ancestor once possessed such an appendage since this appendage, in some insects, still develops as a transitory rudimentary structure. Therefore, a mere numerical comparison of segmental appendages is not possible; one has to carefully examine the and the adult structure in order to correctly compare segmental appendages between the groups of the Arthropoda. 2. Ultrastructural investigation of the hypocerebral organ in Onychophora, and comparison to the corpora allata of insects: and in arthropods are under hormonal control and glands that secrete juvenile hormone and ecdysteroids has been identified in insects, crustaceans, and possibly also in chelicerates (Gilbert et al., 2000). Ecdysteroids have been reported in various tissues in onychophorans (Nielsen, 2001) but no gland has been identified that could be secreting them. As the Onychophora is a member of the Arthropoda, and reproduction and moulting obviously are shared characters, it would seem likely that the physiological background to e.g. moulting and reproduction is homologous. Thus, it is likely that homologous glands or tissues secreting hormones that control this are present within the Arthropoda, and if the Ecdysozoa hypothesis is correct, perhaps also among all of the ecdysozoans. Pflugfelder (1948) proposed that the so-called hypocerebral organ of onychophorans is a corpus allatum (the gland in insects responsible for juvenile hormone secretion) analogue, based on its histology. However, no ultrastructural investigation has been done on the hypocerebral organ of the Onychophora. Therefore, it was regarded as important to do such an investigation in order to acquire additional data for making a judgment of the function of the hypocerebral organ and its possible homologues within the Arthropoda. 3. Expression pattern of the gene engrailed: Evolution of morphology is evolution of development and during the last decades, genetic expression and function has emerged as an additional tool to study evolution. It is not easy to directly connect gene expression with morphology unless it is possible to construct knock-outs or knock-downs. This is often difficult to achieve in non-model organisms, but nevertheless, by comparing the expression of certain developmentally important genes in non-model organisms with expression in model organisms where a mutant phenotype has been documented might still provide important information. The segment polarity gene engrailed has been used as a segmental marker and has also been demonstrated to be important for nerve cell development. Since earlier investigations have not demonstrated expression in the brain-

22 antennal segment or in the developing nervous system it was decided that additional tests had to be conducted with more developmental stages examined than before to see if a broader expression pattern could be detected. The expression in the brain-antennal segment would be particularly useful to re-investigate since this could provide information as to how many segments this area of the embryo contains.

Methodology

During the course of this investigation a number of techniques have been used in order to visualize morphology and gene expression. A brief summary of methods is given below. For more detailed descriptions, please refer to the actual papers and manuscripts.

Detection and description of cephalic nerves Adult onychophoran heads were embedded in either paraffin or epoxy resin and then sectioned. Staining was accomplished with methylene blue for epoxy embedded specimens and with Mallory’s trichromatic stain according to Ladewig for paraffin embedded specimens. Mounted sections were analysed in a conventional light microscope. Cut edges of antennae were back-stained with DiI in order to detect nerve fibre tracts in the brain. DiI is a lipid-soluble crystalline compound (1,1’- dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanineperchlorate) that migrates in the cell-membrane. Hence, it will preferentially follow the membrane of those axons were it was placed, and not easily be taken up by neighbouring cells. Crystals of DiI were put on the cut edges of the antennae for about a month, and then analysed in a fluorescent stereo microscope. This technique was only possible to apply on the relatively large antennal nerves, since application of the DiI crystals was done by hand using no other help than a pair of forceps and a stereo microscope. Immunohistochemical staining was performed in order to obtain a three dimensional view of the cephalic nerves and associated appendages. A monoclonal antibody directed towards acetylated α-tubulin (a protein specific for nerve fibres) was used together with a secondary antibody coupled to either a fluorochrome or horse-radish peroxidase (HRP). Embryos treated with a fluorochromatic secondary antibody were analysed in a Confocal Laser Scanning Microscope (CLSM) and image stacks were combined to form a three dimensional image. The HRP-coupled secondary antibody was used on both whole-mounts and on sections. HRP is an enzyme that in the presence of hydrogen peroxide transforms an added

23 substrate (di-amino-benzidine) to a brown precipitate. Specimens treated with HRP-coupled secondary antibodies were analysed in a light- or stereo- microscope.

Embryological investigations During the embryological investigations the above mentioned techniques were used, as well as Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) in order to investigate the ultrastructure. It was decided to use in situ hybridisation to analyse the expression of the gene engrailed. A pair of degenerate primers was designed and polymerase chain reaction (PCR) under low stringency conditions yielded a 350 base pair long sequence of engrailed. This sequence was used to design specific primers that yielded a 250 base pair long sequence that was labelled with dioxygenin (DIG) in a PCR reaction.The in situ reaction was carried out according to established protocols (see paper IV) and then incubated with fragmented antibodies directed towards DIG and coupled to alkaline phosphatase. After extensive rinsing steps, a colour reaction was achieved by adding a substrate for the alkaline phosphatase, and the staining was stopped when sufficient coloration was detected. The stained embryos were analysed as whole-mounts in a stereo microscope, or embedded in paraffin, sectioned and analysed in a light microscope.

24 Results and discussion

Innervation of the head (paper I) The first paper in this thesis, Onychophoran cephalic nerves and their bearing on our understanding of head segmentation and stem-group evolution of Arthropoda, presents a new scheme for the innervation of the onychophoran head, which differs significantly from previous investigations. The innervation was investigated with the help of classical histological techniques carried out on adults as well as with immunohistochemical staining of whole-mounted embryos that were analysed in a confocal laser scanning microscope. These two independent techniques gave identical results, and, hence, we can be certain that the innervation scheme presented in this paper is more correct than those presented in previous investigations. The data presented in this paper confirm that the so-called labrum in onychophorans is a part of the pharynx and affinities to any pre-oral outgrowths in arthropods such as the labrum can be ruled out. It further presents evidence for a terminally placed mouth in the onychophoran ancestor, and that the ventralisation of the mouth in the line leading to arthropods may have been a parallel event. The innervation pattern presented in the paper can be interpreted as a modified circumoral nerve ring similar to that seen in cycloneuralians, and this, together with the evidence for a terminally placed mouth in a basal onychophoran, supports the Ecdysozoa hypothesis.

Development of the nervous system (paper II) The second paper, Head Development in the Onychophoran Euperipatoides kanangrensis with Particular Reference to the Central Nervous System, is an investigation of the head development in an onychophoran. The data presented show that the onychophoran antenna develops in front of the eye in the most anterior part of the brain; therefore it is not regarded as a homologue to the arthropod first antenna but more likely to the arthropod labrum. The paper presents further evidence for an 25 originally terminal mouth in the onychophoran stem-lineage. The two most anterior oral lips originate in a dorsal position and later during development migrate to their final ventral position. These oral lips are also innervated in the adult from a dorsal part of the brain (data presented in the first paper), which corroborates the idea of an originally terminal mouth. In spiders, insects and crustaceans, neuroectoderm produces at discrete locations and in discrete time waves (Anderson, 1973; Scholtz, 1992; Stollewerk et al., 2001). In this paper it is confirmed that this is not the case in onychophorans, where the neuroectoderm produces precursor cells all over its surface during a continous and prolonged time-period. This non- generalised way of nerve cell production may turn out to be basal, and the more specified nerve cell production seen in the euarthropods could have evolved in concert with the evolution of highly specialised tagmata.

The hypocerebral organ (paper III) The third paper, An Ultrastructural Investigation of the Hypocerebral Organ of the Adult Euperipatoides kanangrensis (Onychophora, Peripatopsidae), describes the ultrastructure of the hypocerebral organ and confirm that it is, most likely, a gland. However, the corpus allatum of insects is a neurosecretory gland that is innervated from the brain, and no such innervations were found in the hypocerebral organ. This makes homologisation difficult. However, neurosecretory cells were discovered in the brain in close association to the hypocerebral organ and cellular strands connecting the brain with the hypocerebral organ were found to be present. These cellular strands could represent an alternative brain-hypocerebral organ communication pathway. Even if the hypocerebral organ most likely is a gland of some kind, it is impossible with the present data to assign an equivalent within the Arthropoda. Nevertheless, it is important to investigate further this large and conspicuous organ which may prove to add vital information to the evolution of arthropod endocrinology.

Expression of the gene engrailed during development (paper IV) The expression of engrailed was investigated for different developmental stages; before segmentation, just after the onset of segmentation, just after neurogenesis had started in the brain anlage, and in near completed embryos. The first signs of expression were detected in embryos were neurogenesis had started. These embryos showed expression in a subset of neuronal precursors located in the brain anlage and in the growing tip of the limb 26 buds. The engrailed positive cells of the brain anlage were located at an area where the first commissure will form. This might indicate that engrailed has a function as an axon guidance cue and/or as a neuronal identity marker, as has been described for other animals before (Marie et al., 2002; McDonald and Doe, 1997). The expression in the growing limb buds was detected both in ectodermal and mesodermal cells. Patterning of the limb buds by engrailed has been described in the mouse (Loomis et al., 1996) and expression of engrailed has also been reported to occur where sclerotisation occurs (Jacobs et al., 2000). It is not possible with the prevailing data to connect either of these two functions to engrailed expression seen in onychophorans, but they certainly provide possible explanations.

Conclusions • A new scheme of the innervation of cephalic structures and appendages is presented. The innervation-pattern of the mouth is interesting in that it suggests that a terminal mouth was a basal character of the onychophora, and furthermore it indicates that the ventralisation of the mouth in onychophora and euarthropoda may have been parallel events. These data are interpreted as supportive to the Ecdysozoa hypothesis. • The developmental investigation gives further support for a ventrally placed mouth as a basal character of the Onychophora. This, taken together with the fact that fossil members of the arthropod stem group possessed a terminal mouth, is indicative of the absence of an acron in the groundplan of the Arthropoda. The developmental data also point to the fact that the eye and the antenna of onychophorans belong to the same segment. This segment is the most anterior one in onychophorans and if homologous seriality applies to the Arthropoda, this would suggest that the antenna of onychophorans is homologous to an appendage of the anteriormost segment of euarthropods. A possible candidate to such an appendage within euarthropods is the labrum, but the true nature of the labrum of euarthropods needs to be clarified in order for such a proposal to be confirmed. • Ultrastructural investigation of the hypocerebral organ confirmed that this structure is glandular with epithelial-like cell junctions facing the lumen. No innervation was found to be present but cellular strands connect the hypocerebral organ with the brain, which could serve as communication between the brain and the hypocerebral organ.

27 • The expression pattern of engrailed was found to be different from earlier accounts in Onychophora. The expression in the developing brain suggests that this engrailed paralogue functions as an axon guidance cue, which has been demonstrated in other arthropods. The present data, together with previous gene expression data, therefore imply that that there are at least two engrailed paralogues in onychophorans with split functions that normally are shared by one engrailed paralogue in other organisms.

28 Acknowledgements

I wish to thank the following people for invaluable help during the course of this investigation: The staff of the Department of Evolutionary Biology Centre, Programme of Animal Development and Genetics; Jan Löfberg, Lennart Olsson, Rolf Ericsson, Rolf Olsson, Claes Holmgren, Helena Malmikumpu, Gun-Britt Örlander, Åke Franzén, Chandrasekhar Kanduri, Meena Kanduri, Joanne Whitehead, Vinod Pant, Anita Mattsson, Vasudeva Ginjala, Noopur Thakur, Magda Lezcano, Piero Mariano, Rituparna Mukhopadhyay, Yu Wenqiang, Carl-Eric Cantell, Carina Östman, Erik Ullerås, Marie Arvola. Persons affiliated with other programmes of the Evolutionary Biology Centre: Stefan Gunnarsson, Anette Axén, Gary Wife, Per-Owe Thörnqvist, Svante Winberg, Mats Block, Anbar Khodabandeh, Karin, Gunnar Andersson, Tove Andren,Olivier Lepage, Svante Winberg, Kenneth Söderhäll. The staff of the Programme of Historical Geology and Palaeontology: John Peel, Lars Holmer, Ulf Sturesson, Birgit Jansson, Gudrun Söderman, Malgorzata Moczydlowska-Vidal, Enrico Savazzi, Joakim Samuelsson, Martin Stockfors, Gerard Malsher, Vivianne Berg- Madsen (presently at the Museum of Evolution), Solweig Stuenes (presently at the Museum of Evolution), Jenny Sälgeback, Christian Skovstedt, Chris Atkins, Ole Hoel, Anna Lindström, Eva Egerqvist, Alexander Gubanov, Jan-Ove Ebbestad, Anette Högström, Henning Blom. My supervisors Graham Budd and John Peel are thanked for much support during these years. Noel Tait is thanked for his invaluable help as regards to collecting of onychophorans, hospitality in Sydney, constructive criticism of manuscripts, and acting as a general source of inspiration. Debra Birch and Jenny Norman are greatly acknowledged for their help with microscopy preparations in Sydney. Earl Larson and Per-Owe Thörnqvist are warmly thanked for much help with the molecular side of

29 the project. Gerard Malsher is thanked for fruitful discussions at the Klubban biological station. The personnel at Klubban biological station are thanked for help in searching for pycnogonids and other organisms. Petter Melin, Martin Stockfors and Jonas Ahnesjö have been prolific sources of scientific and recreational inspiration. My is greatly acknowledged for providing the substantial support that has made the completion of this task possible.

30 References

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