Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 848

A Comparative Study of Head Development in Mexican Axolotl and Australian Lungfish: Cell Migration, Cell Fate and Morphogenesis

BY ROLF ERICSSON

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3--5 !! 17 > 3-45 6!7221722172  "  """ 7 111 9 "?? &<&? @ A "  """ 7 111: This thesis is based on the following papers, which will be referred to by their Roman numerals:

I Ericsson R., Olsson L. (2003) Patterns of Spatial and Temporal Visceral Arch Muscle Development in the Mexican Axolotl (Ambystoma mexicanum) (Accepted for publication in Journal of Morphology)

II Ericsson R., Cerny R., Falck P., Olsson L. (2003) The Role of Cranial Neural Crest Cells in Visceral Arch Muscle Positioning and Patterning in the Mexican axolotl, Ambystoma mexicanum. (Manuscript)

III Cerny R., Ericsson R., Meulemans D., Bronner-Fraser M., Epperlein H-H. (2003) Mandibular arch morphogenesis and the origin of jaws: what does “maxillary” and “mandibulary” really mean? (Manuscript)

IV Ericsson R., Joss J., Olsson L. (2003) Patterns of Spatial and Temporal Visceral Arch Muscle Development in the Australian lungfish, Neoceratodus forsteri. (Manuscript)

Contents

Introduction...... 6 Origin of jaws ...... 10 Origin of tetrapods...... 11 The segmented head...... 12 Hindbrain segmentation...... 12 Retinoic acid ...... 12 Hox genes ...... 13 Eph receptors and ephrin ligands...... 15 Developmental impact of hindbrain segmentation ...... 16 Ectoderm ...... 16 Endoderm...... 17 Mesoderm...... 18 Development...... 18 Myogenesis ...... 19 Osteogenesis ...... 20 Angiogenesis...... 21 The neural crest ...... 22 History ...... 22 Development...... 23 Induction ...... 23 Migration...... 24 Differentiation ...... 26 Development and evolution ...... 27 Aims of the present investigation...... 28 Results and discussion...... 29 Acknowledgement ...... 34 References ...... 35 Introduction

The vertebrate head is a complex and unique structure, which consists of a complicated mixture of organs and tissues. Even given this apparent complexity, there are hidden borders and patterns. The layout of the head starts early in development and includes a wide range of cell-cell interactions and complex expression patterns of a multitude of molecules. The focus of interest for most developmental biologists working with the head is some transient structures in the hindbrain, the rhombomeres (Fig. 1). These are serially arranged swellings, the numbers varying among species from seven to eight. Each of these swellings has its own identity, determined by the expression pattern of a special group of genes, the Hox-genes. The identity of the rhombomere is kept in the daughter cells throughout development. On top of the developing neural tube, underneath the ectoderm, a new embryonic organ forms, the neural crest. The neural crest cells are unique to vertebrates and are considered to be crucial in the evolution of the vertebrate head. These cells will migrate away from the neural tube and give rise to a large variety of tissues and cell types (Table 1). In the head the identity of the neural crest cells is determined by the rhombomere from which they exit. The borders of the rhombomeres are borders for the neural crest cells too, and they will only migrate laterally away from the hindbrain. However, they do not migrate as a continuous sheet of cells but rather in separate streams. The first two rhombomeres give rise to the mandibular stream of neural crest cells, the fourth rhombomere to the hyoid stream and the sixth and seventh (and eighth, if there is one) to the branchial streams of neural crest cells. There is no migration lateral to the third and fifth rhombomere. The neural crest cells from these areas join the cell streams emerging from the neighbouring rhombomeres. The streams of cells migrate laterally and ventrally between the overlying ectoderm and underlying mesoderm and form the embryonic structures called the visceral arches. The mandibular stream ends up in the mandibular arch, the hyoid stream in the hyoid arch and the branchial streams in the branchial arches. In the visceral arches the neural crest cells encircle the underlying mesoderm to form a tube, with mesoderm in the middle. The medial part of each visceral arch is covered with endoderm and the lateral part with ectoderm. Each arch will give rise to a specific set of structures. Investigations performed mostly on chicken and

6 Figure 1. Schematic presentation of the segmentation of different tissues in the head region (ectoderm omitted) of an amniote embryo at the visceral arch stage. The dashed lines delineate the visceral arches. r1-r7, rhombomeres; s1-s5, somites.(Modified from 1)

7 quail embryos have given rise to the following image of cranial neural crest and mesoderm development: Neural crest cells of the mandibular arch give rise to the upper and lower jaws and connective tissue for the masticatory muscles. The mesoderm of the mandibular arch gives rise to the masticatory muscles. The neural crest cells of the hyoid arch form the bones of the hyoid apparatus and the connective tissues associated with these muscles. The mesoderm of the hyoid arch forms the muscles associated with these bones.

Table 1. A list of some of the derivatives of the neural crest cells, and of tissues and organs containing cells that are derived from the neural crest. (Adapted from 2)

Cell types Sensory neurons Rohon-Béard cells Satellite cells Schwann cells Glial cells Calcitonin-producing cells Melanocytes Chondroblasts, chondrocytes Osteoblasts, osteocytes Odontoblasts Fibroblasts Mesenchymal cells Striated myoblasts Smooth myoblasts Angioblasts Adipocytes

Tissues and organs Spinal ganglia Peripheral nervous system Thyroid gland Adrenal gland Craniofacial skeleton Dentine Connective tissue Adipose tissue Smooth muscles Striated muscles Cardiac septa Dermis

In the branchial arches, neural crest cells form the bones and the connective tissue of the branchial apparatus or the gill cage (in animals with gills) and the mesoderm, subsequently, give rise to the associated muscles. In short, the muscles of one visceral arch attach to bones derived from neural crest cells of the same arch and are innervated by nerves originating from the same axial level as the neural crest cells of the same arch. The pattern originating

8 from the rhombomeres in the hindbrain is maintained, even though the head now does not look segmented at all. This patterning mechanism seems to apply to head development in all vertebrates, and must therefore be as old as the vertebrate clade itself. Despite this common mechanism though, vertebrates have a highly variable head morphology. How have developmental mechanisms evolved to generate this vast variety? The focus of this study has been to look at if and how the evolution of terrestrial tetrapods from lobe-finned fish ancestors has affected the development of the head.

Figure 2. Simplified phylogeny of the chordates including the taxa discussed in this thesis.

9 Origin of jaws

The first jawed vertebrates or gnathostomes appeared in the Silurian (408- 439 mya) (Fig. 2). Very little is still known about the ancestors of gnathostomes as the fossil record does not reveal much about this issue 3. The current hypotheses are based mainly on comparative embryological data from extant species. The classical view of the origin of the jaws of gnathostomes is that they are derived from modified gill arches (Fig. 3). Both jawless vertebrates and gnathostomes have gills but only gnathostomes have jaws. Jaws and the gill skeleton are also derived from the same cell type, the neural crest cells. The morphology of the jaws is also very similar to that of the gills. However, there is no instance of craniates with gill- bearing mandibular arches in the fossil record, and there is no evidence from gnathostome development that indicates a mandibular gill. Another hypothesis is that the jaws are derived from a velum-like structure. The velum is a pumping structure in the ammocoetes larva of lampreys, and may have been present in the agnathan ancestor of gnathostomes. It may be derived from the mandibular arch neural crest cells. In this scenario, the jaws would have come first and the organisation of the rest of the visceral arches would have been developed as a support for the jaws. This hypothesis has some support from molecular and migration studies of neural crest cells in lamprey 4,5.

Figure 3. The branchial arch hypothesis of jaw origin. At first, all visceral arches carry gills. Then a joint is formed between the upper and lower parts of the bony elements of the first arch (black) and finally, the second arch (striped) develops as support for the first arch. The remnant of the gill slit between the first two arches forms a spiracle behind the eye (Modified from 3)

10 Origin of tetrapods

The tetrapods belong to a group called the Sarcopterygii, which means “fleshy fins”. They are characterised by having a monobasal articulation of the paired fins with musculature at the base. The earliest known sarcopterygians are Early Devonian (~400 mya) dipnomorphs, i.e. lungfishes and their relatives 6. Of this group, only three genera remain today, Neoceratodus in Australia, Protopterus in Africa and Lepidosiren in South America. Actinistians, to which the coelocanth Latimeria belongs, appear somewhat later, in the Late Middle Devonian (~380 mya). The tetrapods evolved in the Late Devonian (~370 mya) from a group of sarcopterygian fishes called osteolepiformes 7,8 and are characterised by having the paired fins transformed into limbs for locomotion, the presence of the stapes bone in a middle ear structure and a separation of the pectoral girdle from the skull. The first tetrapods were so-called amphibians such as Acanthostega and Ichthyostega. They probably had a mainly aquatic life-style and had large compact skulls. The group to which the modern amphibians belong, the Lissamphibia, probably appeared in the Triassic (290-250 mya) 9. Modern amphibians are divided into three groups, Anura (frogs), Urodela (salamanders) and Gymnophiona (caecilians). The earliest known crown group salamander is from the Middle Jurassic (~230 mya) and belongs to the family Cryptobranchidae 10, which is considered a fairly basal group of salamanders. The Ambystomatidae, to which Ambystoma mexicanum belongs is a modern group of salamanders. The oldest fossils of this family are from the Oligocene period (35-56 mya) 11. Limbs have sometimes been lost, for example in some lizards, in snakes and in caecilians. Even though limbs might not have evolved primarily for terrestrial life, they were more or less a prerequisite for that lifestyle. Limbs are useful for more reasons than locomotion. Defying gravity is a greater problem on land than in the water. Lowering the jaw is no problem for a fish in water, but on land, the head needs to be elevated for the jaw not to hit the ground. For this reason, the head cannot be too heavy or directly attached to the limbs. During evolution, the tetrapods have lost or fused many of the bones that were found in their fish-like ancestors, which has lead to less heavy skulls with new attachment sites for the cranial muscles. The separation of the shoulder girdle from the skull and the appearance of a neck took place early in tetrapod evolution, this separation being already present in Acanthostega. Unfortunately, the fossil record reveals nothing about the developmental processes that have caused these evolutionary changes.

11 The segmented head

The development of the vertebrate head requires contributions from all germ layers in a complex network of interactions that has evolved for hundreds of millions of years. A common developmental feature in bilaterians is the presence of segments or metameres, a sequence of repeated structures. Segments are chacterised by having a positional and molecular identity that will be transferred to the daughter cells, thereby ensuring the development of a certain structure in the correct place. In vertebrate head development, segmentation appears in the brain, in the pharynx and in the visceral arches. A segmentation of the anterior end of the body is a feature not only of vertebrates, but is present in all chordates. In cephalochordates and urochordates, the perforated pharynx is present although the water and food transport is mediated by ciliary movement rather than by muscular pumping 12. Unlike in vertebrates, there is no bone or cartilage support in the pharynx, and there are no aortic arches 13. The central nervous system of the cephalochordates have an anterior cephalic vesicle proposed to be homologous to the vertebrate diencephalon, and a nerve chord expressing Hox genes in a pattern similar to that of vertebrates, although there are no structures similar to rhombomeres 14-18. Gans and Northcutt 13 proposed that everything rostral to the notochord in vertebrates is a “new head”, that has been added through modifications of extant structures and cell types. Indeed, most of the brain and head lies rostral to the tip of the notochord. A revised view of the “new head hypothesis” can be found in Northcutt´s 1996 review 19.

Hindbrain segmentation The brain of vertebrates is divided into distinct parts. The prosencephalon (forebrain) which gives rise to the telencephalon and diencephalon, the mesencephalon (midbrain) and the rhombencephalon (hindbrain) which divides into the metencephalon and myelencephalon. As mentioned above, the hindbrain is transiently segmented into a number of rhombomeres, each of which has an intrinsic identity.

Retinoic acid The anterior-posterior axis of the hindbrain is hypothesised to be controlled by a gradient of Wnts, Fgfs and retinoic acid during early neurulation 20-22. Of these molecules, retinoic acid is the most studied. Retinoic acid (RA) is the biologically active metabolite of vitamine A. It acts on gene transcription

12 through a number of receptors, retinoic acid receptors (RAR) and retinoid X receptors (RXR). In the head, RA is present in a concentration gradient in the paraxial mesoderm that ends at the midbrain-hindbrain junction 23 and has a posteriorising effect on the hindbrain, probably due to the presence of retinoic acid response elements in the enhancers of several Hox genes 23,24. Another pathway could be that RA acts via the expression of Fgfs and Cdx genes to set up the rostro-caudal expression limits of Hox genes 25. Saturating the embryo with RA will cause a spreading of Hoxb1 expression anteriorly and thereby change the identity of rhombomere r2/r3 into that of r4/r5 23,26-29. RA-deficiency in quail embryos leads to lack of rhombomere r4-r8, or at least lack of molecular and morphological specification of that region 30. The regulation of Hox genes by RA is not only a vertebrate feature, but is also present in amphioxus and ascidians, thereby indicating the evolutionary importance of the system 18,31.

Hox genes The rhombomere borders coincide with the borders of expression of several Hox genes 32-35. The genes in the vertebrate Hox gene family share a highly conserved nucleotide sequence of 180 bases with the HOM-C genes in Drosophila 36. The HOM-C genes are segment identity transcription factors and may cause homeotic transformations if expressed in the wrong place, e.g. a leg in the place of an antenna. The HOM-C genes are divided into two clusters in the Drosophila genome 37. In vertebrates, there are four clusters of Hox genes, each cluster containing homologues to both Drosophila clusters 38 (Fig. 4). The position of the single genes on the 3’ to 5’ axis dictates their rostro-caudal and temporal order of expression. The 3’-most gene in each cluster has the rostral-most expression border and is expressed first, with some exceptions 39.

However, Hox genes are not expressed in the entire neural tube. No expression has been detected rostral to the midbrain-hindbrain junction. Hox gene expression is retained in the neural crest cells which arise from the rhombomeres 35,41. The only exception is rhombomere 2, which does not produce Hoxa-2-expressing neural crest cells 42. Knocking out Hox genes causes several different malformations. Hoxa-1-/- mouse embryos have only five rhombomere-like structures, r1–r5 33,43,44. The motor neurons of the abducens nerve are absent and those of the facialis nerve are scarce. If Hoxa- 1 is overexpressed in zebrafish, the mandibular arch cartilages do not form and the hyoid arch cartilages seem to consist of a fusion of the mandibular and hyoid arches 45. A similar pattern was observed when injecting Xenopus embryos with Hoxa-2 46. Hoxa-2 enables the transformation of the first arch into second arch identity even after neural crest cell migration into the

13 visceral arch has taken place 46. Overexpressing Hoxa-2 within its normal domain of expression leads to a transformation of the third visceral arch cartilages into elements resembling the second arch cartilages 46. In Hoxa-2-/- mouse embryos, a mirror image duplication of the mandibular arch cartilages appeared in place of the hyoid arch 47,48. This pattern also appears when transplanting the midbrain-hindbrain isthmus into the position of rhombomere 4 49.

Figure 4. Schematic presentation of the Drosophila HOM-C genes and their vertebrate homologues, the Hox genes of the mouse. The paralogous groups indicate the relationship between the genes of different clusters. The 3’-genes are expressed first and have the anterior-most expression domain. Retinoic acid (RA), mainly affects the Hox-genes of the first 4 paralogous groups, those with expression domains in the head. (Modified from 30,40)

The plasticity of Hox gene expression in the hindbrain has been addressed in a number of studies. When transplanting rhombomeres to the trunk, the Hox gene expression in the transplant is conserved for several days, but will gradually change and become the same as in the neighbouring tissue 50,51. Rotation of the hindbrain give a surprisingly varied result depending on which part of the hindbrain is rotated. Rotation of r1–r7 changes the Hox gene expression in the rotated part to match the new location, while r3–r7 rotation does not change the expression 52. In the migrating neural crest cells Hox gene expression is dependent upon cell-cell interactions. When

14 transplanting clusters of neural crest cells from one migrating stream to another, the original Hox gene expression is retained in the clusters of cells. Cells that split away from the clusters or cells that were injected as a suspension change their Hox gene expression into that of the new environment 53. Hox genes are not only expressed in the hindbrain and neural crest but also in the ectoderm, mesoderm and endoderm of the head (Fig. 5)

Figure 5. Schematic presentation of the expression pattern of Hox genes of the rostral-most paralogous subgroups in different tissues of the head of an embryonic chick. R1-R7, rhombomeres; VA1-VA5, visceral arches (Modified from 30,54)

Eph receptors and ephrin ligands Hox genes are not the only genes that are required for hindbrain patterning. The formation of boundaries between the rhombomeres in the hindbrain is partly due to the sequential expression of Eph receptors and their ligands, the ephrins 55. Eph receptors and ephrin ligands are members of the receptor tyrosine kinase (RTK) family of membrane-bound proteins and may function as repellent cell-surface molecules. Epha4 is expressed at early stages in the presumptive odd-numbered rhombomeres r3 and r5, and a ligand, ephrin-B2 is expressed in the presumptive even-numbered rhombomeres r2, r4 and r6 56-62. The expression of Epha4 is controlled by krox20, which is expressed in the same region 63. Cells expressing the ligand will repel cells expressing the

15 receptor and vice versa, thereby sorting the cells and generating the borders between the rhombomeres.

Developmental impact of hindbrain segmentation The pattern outlined by the rhombomeres and the expression of regulatory genes in the hindbrain is kept throughout development. The branchiomotor nerves innervating the visceral arches exit from the even-numbered rhombomeres and r7 32-34,64,65 (Fig. 6). Trigeminus (V) innervates the mandibular arch muscles and exits from r2, facialis (VII) innervates the hyoid arch muscles and exits from r4, glossopharyngeus (IX) innervates the first branchial arch and exits from r6, and vagus (X) which innervates the muscles of the second and third branchial arches exits from r7. The neural crest cells which migrate away from the same rhombomeres and cells from the neighbouring neurogenic placodes contribute to the sensory neurons of the ganglia of the nerves mentioned above 30,66-69. Disturbing hindbrain segmentation leads to severe malformations, indicating its central role for the development of not only neural tissues but for the development of the head as a whole.

Figure 6. Schematic view of cranial nerves and their motor neurons in the hindbrain. Nerves V, VII and IX innervate the mandibular, hyoid and first branchial arches, respectively. The rest of the branchial arches are innervated by X. (Modified from70)

Ectoderm The presence of a Hox gene expression pattern in the ectoderm, similar to that of the underlying neural tube, led to the hypothesis that the ectoderm is 16 segmented into ectomeres 71. However, transplantations and extirpations have not revealed any developmental significance of this pattern 72. After the onset of neural crest cell migration, placodes form in distinct areas of the ectoderm. The placodes are ectodermal thickenings which give rise to cells in the sensory ganglia of the visceral arch region, the lateral line system, the olfactory epithelium and sensory cells of the inner ear (reviewed in 1,19,30,73). The oral ectoderm produces ameloblasts, which together with neural crest derived odontoblasts are needed for tooth formation (reviewed in 74).

Endoderm The inside of the pharynx is covered by endoderm that has an anterior- posterior regionalisation. The thyroid is derived from endoderm of the second pharyngeal pouch, the thymus from the third pouch and the parathyroid from the fourth pouch 75 76. In zebrafish, the endoderm of the pharynx is specified early by the vgo (van gogh) gene. Mutants lacking vgo do not form pharyngeal pouches and have defects in the pharyngeal cartilages and muscles 77. The endoderm also displays regionalised expression of Hox genes (Fig. 5). Hox genes are not the only genes with regionalised expression in the pharyngeal endoderm. Several signalling molecules are expressed at specific sites in the pharyngeal pouches. Pax1 is expressed in the dorsal tip of the first three pouches, Fgf8 is expressed in the posterior part of each arch, Bmp7 is expressed in the dorsal tip of each pouch and in the posterior part of the second and third arches. Finally, Shh is expressed in the posterior part of the second and third arches 78-80. This regionalisation is kept in the absence of neural crest cells 78. Extirpating parts of the pharyngeal endoderm leads to absence of visceral arch cartilages 72,81. Couly and co-workers 72 showed in an elegant study, that the pharyngeal endoderm patterns the cartilages of the non-Hox expressing neural crest of the first arch, but not the expressing neural crest cells of the more posterior arches. By transplanting ectopic stripes of endoderm from different parts of the anterior pharynx, they achieved duplications of different cartilages. They concluded that the pharyngeal endoderm is responsible for the control of size, shape and orientation of the first visceral arch cartilages. The pharyngeal endoderm also induces the ectoderm to produce the branchiomeric placodes dorsal to the pharyngeal pouches 82.

17 Mesoderm

Development The mesoderm of vertebrates forms during gastrulation and may be considered to be divided into five parts: the chordamesoderm which gives rise to the notochord; the paraxial mesoderm which gives rise to most muscles and to the axial skeleton; the intermediate mesoderm which gives rise to the urogenital system; the lateral plate mesoderm which forms the limb skeleton, the heart and the vascular system, and finally the head mesoderm which contributes to blood vessels, bones and muscles in the head (Fig. 7).

Figure 7. Diagram of the outcome of the different parts of the trunk mesoderm. (Modified from 40)

In the trunk, the paraxial mesoderm is divided into somites. Somites bud off the presomitic mesoderm at its rostral end in a very coordinated manner. In chick embryos, a new somite is produced every 90 minutes 83. Somitogenesis has been shown to involve a molecular oscillator, the segmentation clock, which controls the periodic expression of certain genes related to the Notch pathway 84-87. The expression appears as a wave that sweeps across the presomitic mesoderm during the formation of each somite 88,89. The number of waves correlates with the position of the somite along the anterior- posterior axis. The first somite appears caudal to the otic vesicle. In the head,

18 there are no somites or obvious segments in the paraxial mesoderm. However, there have been reports of a variable number of segments, or somitomeres in several vertebrates 90-92. These segments have only been observed using scanning electron microscopy and their existence has been questioned by several studies 88,93,94. The fate maps of cranial paraxial mesoderm that have been constructed in chicken and mouse show that somitomere borders have no effect on cell migration and cell fate 95-98. Transplantation studies have confirmed that the paraxial mesoderm of the head does not carry an intrinsic identity, but can be replaced by paraxial mesoderm from other areas of the head and even from the trunk 95,98. There is also no molecular data similar to that for the somites which support the presence of somitomeres. The segmental clock shows only two waves of expression in the head, indicating the possible presence of a maximum of two segments 88. The contribution of mesoderm cells to the visceral arches is, however, not random. Several studies have shown that the mesodermal contribution to a visceral arch originates from approximately the same axial level as the neural crest cells of the same arch, i.e. the paraxial mesoderm lateral to the region of the hindbrain that gives rise to the mandibular arch neural crest, contributes to the mesoderm of the mandibular arch 95-98. The regionalisation is not as strict as in the hindbrain, as there is a slight overlap between the contributing areas. The cells of the paraxial mesoderm start migrating approximately at the same time as the neural crest cells. At the formation of the visceral arches, the neural crest cells form a tube around the mesoderm core in each arch. From this stage, the mesoderm divides into a dorsal and a ventral half and then starts to differentiate into muscles and other tissues 99,100.

Myogenesis The developing muscles will continue to be surrounded by neural crest cells, which supply the connective tissue that covers the individual visceral arch muscles 101-104. Myogenesis in the head is delayed compared to in the trunk. In the axolotl and in the chicken, the first muscles to appear are the epaxial muscles 105,106. The first visceral arch muscles appear much later, but still before the muscles of the limbs. The position of the muscles have been argued to be determined by neural crest cells 107. However, recent data from axolotl show that the visceral arch muscles appear in their normal positions without the presence of neural crest cells 104. The individual visceral arch muscles develop from undifferentiated muscle anlagen. Muscle precursor cells can be identified early in the visceral arches by the expression of the basic helix-loop-helix (bHLH) transcriptions factors Myf5 and MyoD 95,105. In many cases the expression of Myf5 and MyoD shows that some individual muscles are specified long before it is possible to distinguish them

19 histologically. Another early marker for muscle development is desmin, an intermediate filament protein that is expressed during the condensation of myoblasts 95,106,108. Myosin is first detected during fibre formation. Not all muscles can be identified as individual muscles before the onset of muscle fibre formation. Some muscles form by splitting from other muscles. In the axolotl, all visceral arch muscles form close to their origin and then extend the muscle fibres towards their insertions 106, a process that requires the presence of neural crest cells to create a normal muscle pattern 104. The pattern follows that of cartilage formation, with the distal muscles forming first and the muscles with attachment sites at a medial position forming last 106. In the chicken the pattern is reversed, with the distal-most muscles forming last 105. The jaw levator muscles of the Australian lungfish have simultaneous fibre formation in the whole muscle anlage and the muscles extend from their insertions to their origins. The molecular basis for myogenesis differs between trunk and head 109. In the trunk, the pre-migratory muscle precursor cells express the transcription factors Lbx, Pax7 and Paraxis 110. In the head these transcription factors are only expressed in the paraxial mesoderm at the trigeminal level 109. The myogenic precursor cells of the masticatory muscles express engrailed (En) which plays no role in trunk myogenesis 111-114. Myf5 and MyoD are expressed both in the trunk and in the head but do not seem to be controlled by the same mechanism in head and trunk. The expression of Myf5 has been shown to be controlled by different promoter elements in the head and in the trunk 115-117. Knocking out both Myf5 and MyoD leads to complete absence of skeletal muscles. In mice with null mutations for both Myf5 and Pax3, no trunk muscles form but the head muscles are unaffected 118. In contrast, knocking out the bLHL transcription factors MyoR and capsulin leads to lack of only the masticatory muscles in mouse 119. The difference in control mechanisms between head and trunk myogenesis could indicate that the vertebrate head is a relatively new structure that has been added during chordate evolution.

Osteogenesis There are two major ways of forming bones, intramembraneous ossification and endochondral ossification. Intramembraneous bones are common in the head and form by direct differentiation of neural crest cells into osteoblasts. Endochondral bones form by replacing a cartilage precursor with bone. Most of the skeleton forms this way. In the head, both the chondrocytes that form the precursors to the bone, and the osteocytes that form the actual bone, are derived from neural crest (with some exceptions). In birds the mesodermally derived bones in the head are corpus sphenoidalis, the orbitosphenoid bone and the otic capsule. The mesodermal cells that contribute to these bones

20 originate from the medial part of the paraxial mesoderm in the head 97. The transcription factor Sox9 is expressed in all precursors to chondrogenic cells. Mice with only one copy of the Sox9 gene show hypoplasia in all endochondral bones 120. Sox9 controls the expression of several collagen genes by binding to their enhancers 121-125. Other members of the Sox-gene family, like Sox5 and Sox6 are involved in the later differentiation of chondrocytes.

Angiogenesis The mesoderm is the source of the endothelial cells in the blood vessels. The formation of blood vessels may occur by two different processes: (1) by angiogenesis, the sprouting and elongation of pre-existing vessels or by merging of pre-existing vessels; (2) by vasculogenesis, when free angioblasts form new vessels in situ 126. The first step in the formation of vessels is the induction of mesodermal cells by TGFß, Fgf and Wnt family genes 127. The initial specification of angioblasts is controlled by vascular endothelial growth factor (VEGF) and its receptors, which when knocked out lead to complete absence of blood vessels 128,129. Angioblasts may migrate long distances and take part in vessel formation far from their origin 70. The specification of blood vessels into arteries and veins seems to be partly controlled by the expression of Eph receptors and ephrin ligands. In mouse the arteries express ephrin-B2 and the veins Eph-B4 130. Disrupting ephrin-B2 in mouse does not affect primary angiogenesis but disrupts the remodelling of both veins and arteries. In the head region the first major vessel to appear is the double ventral aorta, which runs from the heart to the brain, where the primitive anterior arches turn dorsally to connect with the dorsal aortae 131. The primitive anterior arches contribute to the first pair of aortic arches. New aortic arches are added caudally to the first arches during development, thereby forming a single ventral aorta between the arches. In lungfish and axolotl, 6 pairs of arches form, which is considered to be the primitive pattern 40. Mammals also share this pattern while sharks may have as many as 12 pairs. In lungfish, the first pair is lost later during development and in axolotl the second pair is lost as well. The aortic arches are derived from cells from the visceral arches: endothelial cells from the mesoderm, smooth muscle cells and connective tissue from neural crest cells. In fishes and larval amphibians, some of the aortic arches later develop into gill arteries.

21 The neural crest

History Ever since its discovery by Wilhelm His in 1868 there has been great controversy about the importance of the neural crest. From being considered as merely a source for cranial and spinal ganglia to being a major player in the evolution of the vertebrate head 13, the road was long and winding. The existence of a neural crest was a violation of the central dogma of the germ- layer theory. The germ-layer theory was widely accepted among anatomists and embryologists around the end of the 19th century. It states that all tissues are derived from three layers that appear during early embryogenesis. The ectoderm gives rise to nerves and epidermis and the endoderm forms the alimentary canal. All bone, connective tissue, muscle and blood vessels were considered to be derived from mesoderm. In the vertebrate head, however, observations did not match theory. The first study to oppose the old idea was that of Julia B Platt in 1893 132. She claimed that the neural crest, or mesectoderm as she called it, contributed to the cartilage of the jaw apparatus in Necturus. This was fiercely opposed by other scientists who considered this to be impossible. When more studies came that verified Platt’s data, like those of Landacre 133 and Stone 134-136, the advocates of the germ layer theory had to abandon or modify it. The studies by Hörstadius and Sellman 81 showed that the neural folds, from which the neural crest cells arise, were in fact regionalised. When extirpations (ablations) of different parts of the neural folds were made in neurula-stage axolotl embryos, different cartilages were affected. They constructed a map of the neurula, showing the origin of the cells that participated in the development of the cranial cartilages. The cartilages of the mandibular arch, such as those of the jaw, originated from the anterior-most neural crest. The cartilages of the hyoid arch, such as the hyoid bones, originated from the area behind the mandibular crest and the cartilages of branchial arches, such as those in the gill cage, originated from the area behind the hyoid arch crest. After this study, several fate maps of neural crest cells have been produced that confirm these results. One of the first methods to produce good fate maps was to use 3H-thymidine to label the neural crest. This technique was exploited in amphibians by Chibon 137 and in chicken by Weston 138 and Johnston 139. The development of the quail-chick chimaera method by Le Douarin 140-143, where quail cells that have been transplanted into chicken can be distinguished in histological sections and later the construction of the quail-chick marker antibody (which binds specifically to quail cells), led to very detailed fate maps of the neural crest. This is still the best method for long-time tracing of cells. Other methods, such as interspecific transplantations and injections of vital dyes, such as DiI, have been used to 22 produce fate maps in other species, though none is comparable to those in chicken. There are a few reports about transgenic mice expressing a reporter gene in all neural crest cells and their derivatives and hopefully there are more to come144,145. The use of mouse genetics will be a powerful tool in the study of neural crest cell development.

Development Neural crest development can be seen as a number of different steps: (1), induction, where the neural crest is specified as neural crest; (2), migration, where the neural crest cells migrate away from the neural tube and into the tissues of the rest of the head and body and (3), differentiation, where the neural crest cells form the tissues and structures we find in the developed organism.

Induction The neural crest provides a classical example of cell induction and is therefore well studied. Most of our knowledge in this field comes from studies of Xenopus, zebrafish and chicken. The process involves tissue interactions in several steps and a complicated use of molecules that may have different functions at different stages. Below is a brief summary of the important events and factors involved. A thorough analysis of gene expression in the neural crest can be found in Gammill and Bronner-Fraser 146. For further reading, see reviews by Knecht and Bronner-Fraser 147, Aybar and Mayor 21 and references therein. The induction of neural crest cells can be seen as a step-wise process:

1. The formation of the neural plate is dependent on the activity of BMPs and BMP antagonists in the ectoderm 21,22,148. 2. The induction of neural crest potential in the neural folds is dependent on the expression of Snail, Wnts, Fgfs and retinoic acid in the epidermis and/or lateral mesoderm 20-22,149-153. These factors also determine the anterior-posterior axis of the neural tube 20,22. 3. After the formation of the neural tube, the maintenance of the neural crest potential is dependent on the expression of BMPs, Wnt1, Wnt3a and Pax3 in the dorsal neural tube 153-157. 4. The separation into neural crest and neural tube cell lineages requires Notch/Delta signalling and Foxd3 158-163. 5. The emigration of neural crest cells from the neural tube is dependent on the expression of BMPs, Slug and Snail 150,164-170. The delamination from the neural tube is correlated with the expression of cadherin-7 and with down-regulation of cell adhesion molecules such as NCAM, cadherin-6 and N-cadherin 171-174.

23 Other factors involved in this process are Gli3, Zics 175,176 and meis1b 177. The timing and usage of the factors listed above varies between species. The areas of expression may also differ between species. Even though this system has been studied for a long time, new factors and pathways are continuously being discovered.

Migration The onset of neural crest migration away from the neural tube varies between species. In marsupials it is very early, long before the closure of the neural tube 178-180, while in many amphibians, it occurs after the closure of the neural tube 181. In chicken and amphibians, neural crest forms a layer of cells on top of the neural tube, beneath the covering ectoderm after delamination from the basal lamina. In the head, they divide up into the mandibular, hyoid and branchial streams of cells that migrate either laterally between the mesoderm and the ectoderm or ventrally along the side of the neural tube (Fig. 8). In the head, the lateral path is predominant.

Figure 8. Schematic diagram of neural crest cells (grey) migrating in the trunk. The crest cells are migrating between the epidermis and the myotome, ventrally towards the notochord and aorta, and through the anterior part of the sclerotome. (Modified from 40)

24 There is no mixing of cells between the mandibular, hyoid and branchial streams. The branchial streams have no clear borders and mix with each other. Apart from the physical borders, such as the pharyngeal pouches and otic vesicle, there are molecular borders that prevent the different streams of cells from meeting. No neural crest cells migrate lateral to rhombomere r3 and r5. The cells from these rhombomeres migrate either into the mandibular, hyoid or branchial neural crest cell streams. Only these two rhombomeres express the transcription factor krox-20 182. When this gene is knocked out, the migration pattern is disrupted 183,184. As already mentioned, krox-20 is regulates the transcription of EphA4 in rhombomeres r3 and r5. EphA4 is crucial for rhombomere boundary formation. It is also expressed in the migrating neural crest and mesoderm cells of the third visceral arch, and EphB1 is expressed in the third and fourth visceral arches. The ligand ephrin-B2 is expressed in the corresponding cells in the second arch, thereby restricting the area of migration for the third arch neural crest cells 185,186. There seems to be different mechanisms controlling the early and late migration of neural crest cells. Decreasing the expression of Fgfr in mouse only affects the later migration of neural crest cells 187. The laterally migrating neural crest cells of the head region enter into the visceral arches, which consist of endoderm, covering the internal part, a tube of neural crest cells with mesoderm in the middle, and ectoderm covering the outer part. The way the neural crest cells migrate to achieve this structure differs between the two species studied. In chicken, a portion of the neural crest cells migrate through the mesoderm at the dorsal end of the future arch and then colonise the area between the mesoderm and the endoderm 105,107. In axolotl, the neural crest cells move in an outward-in movement encircling the mesoderm, after the neural crest cells already have migrated to the ventral part of the future arch. From the cylinder structure, the neural crest cells of the medial side form condensations of cells or bulges where the future cartilages will start to form. In the mandibular arch of the axolotl, there are three such condensations, a ventral condensation that will eventually extend all the way to the ventral midline and form Meckel’s cartilage, a dorsal condensation that will form the palatoquadrate cartilage, attaching the lower jaw to the skull, and finally a maxillary condensation, rostral to the palatoquadrate condensation that will form the trabecular cartilage 188. The migration pattern of neural crest cells in the trunk is different from that of the head. In the chicken embryo, the cells taking the lateral pathway may be delayed up to one day compared to those taking the ventral pathway. The cells migrating laterally give rise to melanocytes in the epidermis. The cells migrating along the ventral pathway split up into several populations. Some of them migrate ventrally between adjacent somites towards the dorsal

25 aorta and give rise to the primary sympathetic ganglia. The other populations migrate either through the sclerotome or between the sclerotome and the dermomyotome in the rostral part of the somite (Fig. 8). The migration through the somite is controlled by the expression of the EphB3 receptor in the neural crest cells and ephrin-B1 ligand in the caudal part of the somite 189,190. A number of extracellular matrix and cell adhesion molecules, e.g. F- spondin, fibronectin, laminin, tenascin and cadherins, have been shown to permit or inhibit neural crest cell migration. A comprehensive review of the different roles of these molecules is given by Krull 191.

Differentiation There is an ongoing discussion about whether the neural crest cells are prespecified or plastic in their differentiation pathways. Recent studies indicate that interactions with neighbouring tissues and cells are important in determining the identity of neural crest cells 192, though in most cases this does not affect the outcome of a single cell, but rather the identity of the structure of which the cells are a part. In an elegant study by Schneider 193, where quail neural crest cells were transplanted into the area of the skeletogenic mesoderm of the head in chicken embryos, the neural crest cells participated in the construction of mesodermally derived bones. In the head, the neural crest cells differentiate into cartilages and connective tissue specific to each visceral arch. All the bones in the head, apart from the corpus sphenoidalis, the orbitosphenoid bone and the otic capsule, are derived from neural crest cells. Apart from these tissues they also form many other cell types and tissues as listed above in Table 1. In the trunk, they do not form cartilage or bone, which are derived from mesoderm. The differentiation of cartilage and bone is described in more detail in the mesoderm section of this thesis. As already mentioned above, the anterior-posterior identity of cranial neural crest cells is determined by the expression of Hox genes. Two recent studies show that the nested expression of Dlx genes is responsible for the proximo-distal patterning of the jaw 194,195. Dlx genes are the vertebrate homologues of the Drosophila distalless gene and are organised in pairs in the genome. In the two most anterior visceral arches, Dlx1&2 are expressed in the entire arches, Dlx5&6 are expressed in the distal halves and Dlx3&7 are expressed in the distal tips 196-198. Knocking out one of the genes in a pair results in only minor defects. In Dlx1/2 double knock-out mice, only the proximal part was deformed 199. Knocking out both Dlx5 and Dlx6 resulted in a homeotic transformation of the lower jaw into an upper jaw phenotype 194,195. The upper jaw had several deformations, but the bones were still recognizable. Almost all other bones in the head were also affected. In all jawed vertebrates apart from sharks and some Paleozoic groups of

26 vertebrates, the upper and lower jaws differ in morphology. The apparent similarity achieved in the knock-out mice led to the assumption that nested Dlx expression was important for the evolution of jaws 194,195,200. This assumption is supported by studies of lampreys, which are jawless vertebrates, and show no nested expression of Dlx-genes 198,201,202.

Development and evolution Neural crest cells are unique to vertebrates. The origin of these cells is an important developmental question, and is also significant for research on the evolution of vertebrates. Their contribution to the head of vertebrates plays an important role in the “new head hypothesis” 13,19,203,204. Studies of cephalochordates indicate the presence of migratory cells at the margins of the neural plate 205,206. However, there are still no reports about cells migrating away from the neural tube in a manner similar to the vertebrate neural crest cells. Several neural crest markers have been cloned from amphioxus, indicating that vertebrate neural crest-specific genes have been co-opted from earlier functions 4,17,206,207. The migration pattern is conserved among vertebrates. The division into mandibular, hyoid and branchial streams of neural crest cells is found in agnathans and gnathostomes. The neural crest cells contribute to the same structures and have more or less the same expression pattern of marker genes in all vertebrates, which indicates the pivotal role of this pattern in creating the outline of the vertebrate head. As morphological variation is based on changes in developmental processes, the changes leading to variation must occur at later stages, i.e. during morphogenesis. Changes in the control of the morphogenesis of the neural crest derived structures of the head is the key to the vast variety in head morphology which we find among vertebrates.

27 Aims of the present investigation

The aims of this study were to increase our knowledge of head development in basal sarcopterygians in order to understand the developmental basis for the evolution of a terrestrial life-style. I was specifically interested in studying the neural crest cells and their interactions with other tissues such as muscles during development. The main questions posed were as follows: x What is the normal pattern of migration and morphogenesis of the cranial neural crest cells in Australian lungfish and Mexican axolotl? x What is the normal pattern of morphogenesis of visceral arch muscles in Australian lungfish and Mexican axolotl? x What is the role of the cranial neural crest cells in visceral arch muscle development? x Can the developmental differences account for the morphological differences?

28 Results and discussion

Patterns of Spatial and Temporal Visceral Arch Muscle Development in the Mexican Axolotl (Ambystoma mexicanum) (Paper I) The embryonic development of cranial muscles has been documented from a large number of animals (reviewed in 99). However, in most cases there is no information about the sequence of when different muscles start developing. The most notable study of cranial muscle development is that by Noden et al. (1999)105 in the chicken embryo. They used in situ probes for Myf5 and MyoD and antibodies against myosin to document the entire development of cranial muscles. They showed that the individual muscles are specified very early in development, long before they can be distinguished histologically. In this article we present the sequence and pattern of development for visceral arch muscles in the Mexican axolotl. Owing to the absence of in situ probes and antibodies for axolotl Myf5 and MyoD, we used antibodies against the intermediate filament protein desmin, which appears between the onset of MyoD and myosin expression. By applying a new method for visualisation, utilising confocal scanning microscopy, we managed to produce a four-dimensional map of visceral arch muscle development. Our data show that most muscles of the visceral arches start developing at the same stage in all arches, a pattern which is common in amphibians 208-210. Amphibians also share with zebrafish, birds and mammals the early development of the levator mandibulae muscles, which in all these taxa are the first mandibular arch muscles to develop 105,211-214. Another interesting finding is that the muscles start differentiating close to their origin and then extend towards their insertions after the onset of fibre formation. The interhyoideus posterior muscle, which was earlier thought to be a subdivision of the interhyoideus 99, was shown to have a separate muscle anlage, distinct from the anlage of the interhyoideus muscle. This study was the basis for the analysis of Paper II.

29 The Role of Cranial Neural Crest Cells in Visceral Arch Muscle Positioning and Patterning in the Mexican axolotl, Ambystoma mexicanum (Paper II) After studying the normal pattern of visceral arch muscle development, we were interested in studying the role of neural crest cells in this system. Earlier studies by Hall 215 and Olsson et al. 103 indicated that neural crest cells are important for the development of the visceral arch muscles, despite not contributing muscle cells, just connective tissue. Neural crest cells have been attributed the role of carrying the segmentation information from the hindbrain, and to have a central role in the development and evolution of the head. The study by Noden 107 indicated that the neural crest cells could affect the patterning of visceral arch muscles. However, in a later study he noted that some muscle primordia can be identified prior to the contact with neural crest cells 73. It has also been shown that visceral arches form in the absence of neural crest 78. In this study we tagged neural crest cells with the lipophilic dye DiI or transplanted green fluorescent protein expressing (GFP) cells into non-green hosts to study the migration and fate of cranial neural crest cells. We also extirpated migrating neural crest cells of single visceral arches from axolotl embryos and examined the resulting visceral arch muscle defects. The results showed that neural crest cells appear in association with the visceral arch muscles in the Mexican axolotl as in other species studied 97,102,107. The muscles of the extirpated embryos appeared in their normal positions, but failed to extend to their normal insertions. Many muscles were also frayed and smaller than normal. We conclude that the neural crest is important, not for the positioning of the visceral arch muscles but for their correct developmental patterning.

Mandibular arch morphogenesis and the origin of jaws: what does “maxillary” and “mandibulary” really mean? (Paper III) Despite the well-studied early migration of neural crest cells in several species and the complete fate map of chicken neural crest, the actual morphogenesis of the head has not been thoroughly studied. In this study, we take neural crest cell migration one step further and study the morphogenesis of the jaws in the Mexican axolotl. The classical view of jaw development states that the upper jaw, or maxilla is formed from a dorsal condensation called the “maxillary” condensation and the lower jaw, or mandible is formed from a ventral condensation called the “mandibular” condensation. By tracing neural crest cell migration with AP-2 and snail in situ probes and FITC-dextran or DiI injections, we show the presence of two subpopulations

30 in the anteriormost, or trigeminal crest cell population: The premandibular and the mandibular crest cells. The two populations are divided by the eye. No cartilage elements were found in the premandibular area at the stages we examined. In the mandibular area however, two condensations are formed, a dorsal, or “maxillary”, ventral to the eye and a ventral, or “mandibular”, at the tip of the mandibular arch. The mandibular condensation divides into two centres, the ventral-most gives rise to Meckel’s cartilage and the dorsal gives rise to the palatoquadrate. In the area of the maxillary condensation, the trabecular cartilages form. Our study shows that the names “maxillary” and “mandibular” condensations do not correctly reflect their developmental outcome. We propose instead the terms “trabecular” and “maxillo- mandibular” for the dorsal and ventral condensations, respectively. This study indicates that our knowledge of head morphogenesis is very poor, even in an animal that has been extensively studied over the last hundred years.

Patterns of Spatial and Temporal Visceral Arch Muscle Development in the Australian lungfish, Neoceratodus forsteri (Paper IV) After studying the visceral arch muscle development of the axolotl, we were interested in comparing with that of the closest living accessible relative to the tetrapods, the Australian lungfish. The development of the cranial muscles has already been described by Edgeworth 99,216, but he only provides a few time points and show no record of when individual muscles appear. This study enhances the resolution of the early visceral arch muscle development in Neoceratodus forsteri. We used whole mount immunostainings for desmin and standard histology to determine the pattern and sequence of muscle development of the first two visceral arches. In contrast to what we have previously reported, the first muscle fibres appear in the hyoid arch muscles, then in the mandibular arch and finally in the branchial arches. Desmin is present in the muscle anlagen long before fibre formation. The first appearance is patchy but at later stages desmin is present in the whole mandibular muscle anlage and the anterior part of the hyoid muscle anlage. In most cases, muscle fibres form at the same time in the entire anlage. The exceptions are musculus intermandibularis and musculus interhyoideus, both ventral muscles that have their insertions at the ventral midline. The “instant fibre formation” is very different from what is observed in axolotl where fibres usually start forming at the origin and then extend towards the insertion 105,106. In lungfish, the muscles instead form close to their insertion and extends towards their origin.

31 Concluding remarks In this study, we trace axolotl neural crest cells through migration and morphogenesis. We show that the general model of vertebrate jaw development does not match jaw development in the axolotl. The upper jaw does not originate from the “maxillary” condensation as currently assumed, but from an unknown condensation, closer to that of the lower jaw. This pattern can explain old as well as more recent observations of muscle development in the absence of neural crest cells. In normal visceral arch muscle development in the axolotl, the muscles start to form at their origins and then extend to their insertions. The neural crest cells have been considered to control the spatial pattern and identity of visceral arch muscles. This study shows that the position of visceral arch muscles is not dependent on the cranial neural crest cells. Neural crest cells are required for the muscles to extend in the right direction and form the correct attachment points and thereby the correct pattern. The study of Australian lungfish visceral arch muscle development shows a different mode of muscle development than in the Mexican axolotl. In lungfish, the muscle fibres seem to form at the same time in the whole muscle anlage. Our preliminary studies of lungfish neural crest migration indicates more or less complete similarity with all other vertebrates studied. There is still a great need for studies of the morphogenesis of the jaw in lungfish as well, though it should be mentioned that lungfish embryos are both rare and very reluctant to reveal any developmental secrets. The study of lungfish visceral arch muscle development is a step towards acquiring enough data to make an evolutionary comparison of the developmental changes that occurred concurrent with the transition to a terrestrial life-style. This kind of comparison is very difficult and has only been attempted a few times before. Although the Australian lungfish may be superficially similar to the Devonian dipnomorphs, it has evolved for hundreds of million years, just like the branch that eventually led to axolotls. Without the fossil record to indicate the actual gradual changes, all comparison will be restricted to the endpoints rather than the process leading to them. Analyses of the fossil ancestors could potentially give clues to the developmental patterns found in extant species. Despite the vast variation in head morphology seen in vertebrates, the early development is very similar. From lamprey to bird, all embryos pass through a stage with visceral arches. Our knowledge of the genetic machinery controlling these early events is based on a few species such as zebrafish, chicken and mouse but there seems to be a high degree of conservation between them. This indicates the importance of these transient embryonic structures for the general layout of the vertebrate head. However, as all morphological variation, (which is the basis for the action of natural 32 selection) is an effect of developmental events, this similarity as such does not reveal much about how the variation evolved. Studying the later stages of development could therefore reveal much about the evolution of morphological variation, and aid in our understanding of evolution as such. Most studies of cranial neural crest cell migration end with the formation of the visceral arches, mainly because of the lack of molecular markers for later development. The beautiful fate-maps of chick neural crest are only showing the start and end-points and not how the neural crest cells behave through morphogenesis. This kind of information is invaluable when studying the segmentation of the head region but does not provide enough resolution for an understanding of morphogenesis. The factors controlling the morphogenesis of bones, cartilages and muscles are not known, although we have a fairly good knowledge of the genetic regulation of the differentiation of these tissues. In many cases we do not know the whole development of the structures we study, but base our knowledge on homologies with other species or other structures. The studies in this thesis indicate that we need to increase our knowledge of basic head development and morphogenesis at the tissue level as well as at the molecular level.

33 Acknowledgement

This work was carried out at the Department of Animal Development and Genetics. I wish to express my gratitude to the following people:

Supervisors: Rolf Ohlsson Lennart Olsson Graham Budd Past and present members of ZUB: Claes, Erik, Marie, Gail, Gary, Steven, Pierre, Arwen, Liang, Yoichiro, Lai, Vasu, Chandra, Meena, Vinod, Ritu, Noopur, Magda, Joanne, Piero, Wenqiang, Junwang, Anita, Anita, Ragnar, Helena, Calle, Carina, Jan, Åke, Rose-Marie, Gun-Britt, The fish-boys. Palaeo-bunch: Joakim, Ged, Ashley. Jena team: Mats, Christian, Katja. Dresden team: Robert, Hans. Sydney team: Jean, Greg, Li, Jenny. BSA: Stefan, Gary, Annette. RA, The Laundbergs, Dr. Carlsbecker, Olle. Funding: Helge Ax:son Johnsons Stiftelse, Stiftelsen Lars Hiertas Minne, Stiftelsen för Zoologisk Forskning, DAAD, Kungliga Vetenskapsakademien Anyone I might have forgotten.

And of course: Kerstin, my family. gutta cavat lápidem non vi sed saepe cadendo

34 References

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