<p>The development of the vertebrate head is a complex process involving interactions between a multitude of cell types and tissues. This thesis describes the development of the cranial neural crest and of the visceral arch muscles in the head of two species. One, the Mexican axolotl (<i>Ambystoma mexicanum</i>), is a basal tetrapod, whereas the other, the Australian lungfish (<i>Neoceratodus forsteri</i>), belongs to the Dipnoi, the extant sister group of the Tetrapoda. </p><p>The migration of neural crest cells, which form most of the bones and connective tissues in the head, and the morphogenesis of the jaw, was determined in the Mexican axolotl. It was shown that both the upper and lower jaws form from ventral condensations of neural crest cells in the mandibular arch. The dorsal condensation, earlier considered to give rise to the upper jaw, was shown to form the trabecula cranii.</p><p>The normal spatio-temporal development of visceral arch muscles was investigated in both the Mexican axolotl and the Australian lungfish. In axolotl, the muscles tended to start forming almost simultaneously in all visceral arches at their future origins and extend towards their future insertions at the onset of muscle fibre formation. In lungfish, fibres formed simultaneously throughout most of each muscle anlage in the first and second visceral arch, but were delayed in the branchial arches. The anlagen were first observed at their future insertion, from which they developed towards future origins. </p><p>To test the ability of neural crest cells to pattern the visceral arch muscles, migrating crest cells were extirpated from axolotl embryos, which resulted in a wide range of muscle malformations. In most cases, the muscles appeared in the right position but were small and extended in abnormal directions. This shows that neural crest cells are responsible not for the position of the muscles but for their correct anatomical pattern. Fate mapping showed that connective tissue surrounding myofibers is, at least partly, neural crest derived.</p><p>In conclusion, the work presented in this thesis shows that although early development may map out the patterns of later development, the differences between axolotl and lungfish head development are not seen until during morphogenesis. Further investigation of morphogenesis is needed to explain the great variation of head morphology seen in vertebrates today.</p>

The development of the vertebrate head is a complex process involving interactions between a multitude of cell types and tissues. This thesis describes the development of the cranial neural crest and of the visceral arch muscles in the head of two species. One, the Mexican axolotl (Ambystoma mexicanum), is a basal tetrapod, whereas the other, the Australian lungfish (Neoceratodus forsteri), belongs to the Dipnoi, the extant sister group of the Tetrapoda. The migration of neural crest cells, which form most of the bones and connective tissues in the head, and the morphogenesis of the jaw, was determined in the Mexican axolotl. It was shown that both the upper and lower jaws form from ventral condensations of neural crest cells in the mandibular arch. The dorsal condensation, earlier considered to give rise to the upper jaw, was shown to form the trabecula cranii. The normal spatio-temporal development of visceral arch muscles was investigated in both the Mexican axolotl and the Australian lungfish. In axolotl, the muscles tended to start forming almost simultaneously in all visceral arches at their future origins and extend towards their future insertions at the onset of muscle fibre formation. In lungfish, fibres formed simultaneously throughout most of each muscle anlage in the first and second visceral arch, but were delayed in the branchial arches. The anlagen were first observed at their future insertion, from which they developed towards future origins. To test the ability of neural crest cells to pattern the visceral arch muscles, migrating crest cells were extirpated from axolotl embryos, which resulted in a wide range of muscle malformations. In most cases, the muscles appeared in the right position but were small and extended in abnormal directions. This shows that neural crest cells are responsible not for the position of the muscles but for their correct anatomical pattern. Fate mapping showed that connective tissue surrounding myofibers is, at least partly, neural crest derived. In conclusion, the work presented in this thesis shows that although early development may map out the patterns of later development, the differences between axolotl and lungfish head development are not seen until during morphogenesis. Further investigation of morphogenesis is needed to explain the great variation of head morphology seen in vertebrates today.

Lepidosirenidae is a clade of freshwater lungfishes that comprise the South American Lepidosiren paradoxa and four African species of the genus Protopterus. These two genera have been geographically separated since the Early Cretaceous break-up of Gondwana, but they share similar biology and skeletal morphology. The lepidosirenid species traditionally were distinguished by a combination of features such as head-to-body ratios, the number of pairs of vertebral ribs, and the presence or absence of external gills, but there are no published discrete skeletal characteristics and no published comparative studies including all extant species. I used High Resolution X-Ray Computed Tomography (CT), X-Ray photography, and alcohol-preserved, cleared-and-stained, and dry skeletal specimens from museum collections to describe the skeletal morphology of all species of lepidosirenid lungfishes in a comparative context. I digitally disarticulated the bones in each CT scan to compile a comprehensive comparative atlas of the cranial and pectoral elements of all extant lungfish. I discovered that the anocleithrum in Lepidosiren paradoxa, which was previously thought to be lacking, is actually present. I also identified skeletal differences between species in the frontoparietal, parasphenoid, supraorbital, and suboperculum. I incorporated those characters into the first morphological phylogenetic analysis to determine the interrelationships of the lepidosirenids. I also used previously published molecular sequence data from the ribosomal RNA gene 16s to run combined morphological and molecular phylogenetic analyses. To generate phylogenetic hypotheses using different types of data and different methods of determining phylogeny, I employed the maximum parsimony, maximum likelihood, and Bayesian inference methods. Lepidosirenidae is monophyletic in almost all analyses, Protopterus is monophyletic in each analysis, and Protopterus annectens and Protopterus aethiopicus are sister taxa in every analysis. The phylogenetic positions of Protopterus dolloi and Protopterus amphibius are incongruent in many of the analyses, which indicates that further examination of the skeletal variation and addition of molecular sequences of different genes is needed. Based on the comparative morphological atlas and the phylogenetic analyses, questions of lepidosirenid biogeography, morphological variation within lungfish, and better identification of lungfish fossils can now be investigated in a more rigorous context. / text

<p> The origin of terrestrial vertebrates from water-dependent fish involved numerous morphological and physiological modifications (Benton, 1990). Interest in the adaptive mechanisms involved in the transition from aquatic to terrestrial environments has led to research involving lungfish. African lungfish are obligatory air breathers and have a primitive lung and characteristically underdeveloped gills compared to freshwater teleosts. The gills are thought to play an important role in CO2 excretion and possibly in water and ionic exchange while in aquatic conditions. At present, little is known about the basic ionoregulatory physiology of lungfishes; the aim of this thesis was to describe the basic principles of ion and water balance in two species of African lungfish, Protopterus dolloi and Protopterus annectens. Patterns and rates are very similar in the two species, apart from differences in water handling at the kidney. In aquatic conditions, plasma ion (Na+, Cl-, Ca2+) levels are lower than in teleost fish. The major site of diffusive water exchange appears to be the gills. The skin is well vascularized and also serves as site of water exchange, and likely Cl- and Ca2+ uptake as well. However, water and ion exchange rates are lower than in freshwater teleosts, probably due to the reduced gill area, though glomerular filtration, urine flow rates (an index of osmotic permeability), and urinary ion excretion rates are comparable to those of teleosts. Water exchange rates increase immediately after feeding, likely associated with specific dynamic action, and decrease with prolonged terrestrialization, likely due to disturbances in gill function. A budget analysis of ion balance indicates that both unidirectional uptake from the water and net uptake from the food (especially for Cl-) are important, whereas unidirectional efflux across the gills and/or skin is a larger route of ion loss than are feces or urine. Despite many physiological differences between freshwater teleosts and the African lungfish, water and ion balance are maintained in a broadly similar fashion and are achieved by compensating for the reduced gill area by ion acquisition via the skin and by greater ion reabsorption by the kidneys.</p> / Thesis / Master of Science (MSc)

Molecular aspects of nitrogen metabolism in vertebrates is an interesting area of physiology and evolution to explore due to the different ways in which animals excrete nitrogenous waste as they transition from an aquatic to a terrestrial lifestyle. Two main products of nitrogen metabolism in fishes are ammonia and urea. Ammonia is produced during protein catabolism and build up of ammonia is toxic. Some aquatic vertebrates convert ammonia into a less toxic compound urea via de novo synthesis through the ornithine-urea cycle (O-UC). Five enzymes are involved in the O-UC: carbamoyl phosphate synthetase (CPS), ornithine carbamoyl transferase (OCT), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (ARG). An accessory enzyme, glutamine synthetase (GS) also participates in the "fish-type" O-UC. Teleosts excrete ammonia passively over their gills into the aquatic environment. The teleost, Opsanus beta, has been shown to increase urea production after 48 hours of crowding. This thesis explored how crowding stress affected nitrogen metabolite levels of ammonia and urea and O-UC gene expression and enzyme activity in O. beta. Lungfishes while in an aquatic environment avoid ammonia toxicity by releasing excess ammonia across their gills, but when stranded on land they produce urea through the O-UC. Urea production via the O-UC has a metabolic cost of at least four ATP molecules. This thesis explored the response of a lungfish, Protopterus annectens, to six days of aerial exposure and re-immersion conditions by measuring concentrations of O-UC mRNA expression and enzyme activity and nitrogen metabolites ammonia and urea. CPS acts as the entry point to the O-UC and based on enzymatic studies, most aquatic vertebrates utilize one isoform of this enzyme (CPSIII) while terrestrial vertebrates utilize a different isoform of this enzyme (CPSI). Lungfishes are a particularly interesting group of air-breathing fishes, not only because of their link to the origins of tetrapods, but also because CPS I may have originated within this group. Both CPS III and CPS I have been enzymatically described within this group. This thesis uses phylogenetics to investigate how CPS nucleotide sequences in lungfishes evolved compared to other vertebrates.

Urodele Amphibien besitzen außergewöhnliche Regenerationsfähigkeiten, die es ihnen ermöglichen, verlorene Körperteile vollständig nachzubilden. Besonders der Axolotl ist aufgrund seiner einfachen Haltung im Labor ein wichtiger Modellorganismus für die Regenerationsforschung. Allerdings schränkt seine pädomorphe Lebensweise – das Ausbleiben einer natürlichen Metamorphose – die Übertragbarkeit der Ergebnisse auf andere Salamanderarten ein. Um Unterschiede und Gemeinsamkeiten in zugrundeliegenden Mechanismen von Regenerationsprozessen zu entschlüsseln sowie evolutionäre und ökologische Einflüsse besser zu verstehen, sind vergleichende Studien mit weiteren Salamanderarten entscheidend. Kapitel I vergleicht die Regenerationsfähigkeit des Axolotls mit der des metamorphosierenden Tigersalamanders. Dabei zeigen larvale Tigersalamander ähnlich beeindruckende Regenerationsfähigkeiten wie Axolotl, während postmetamorphe Individuen eine unvollständige Frakturheilung und verzögerte Regeneration mit Skelettanomalien aufweisen. Kapitel II untersucht die Schwanzregeneration des Tigersalamanders während der Metamorphose und zeigt, dass sie in und auch nach diesem Entwicklungsstadium fortgesetzt wird, obwohl die Geschwindigkeit und die strukturelle Qualität der Regeneration in den verschiedenen Entwicklungsstadien variieren, was den Einfluss der Metamorphose auf den Regenerationsprozess unterstreicht. Kapitel III analysiert die Gliedmaßenregeneration bei sechs plethodontiden Salamanderarten mit verschiedenen Lebensweisen und Habitaten. Alle Arten zeigen erhebliche Regenerationsfähigkeiten, wenngleich häufig anatomische Anomalien auftreten. Zudem werden Korrelationen zwischen der Regenerationsgeschwindigkeit und dem jeweiligen Habitat festgestellt. Kapitel IV untersucht die Flossenregeneration bei Lungenfischen. Dabei auftretende Anomalien ähneln denen von Salamandern und stützen damit die Hypothese, dass epimorphe Regenerationsfähigkeiten einen tiefen evolutiven Ursprung besitzen. / Urodele amphibians possess remarkable regenerative abilities, allowing them to fully restore lost body parts. The axolotl, in particular, has become a key model organism for regeneration research due to its easy maintenance in laboratory settings. However, its paedomorphic life history pattern—the absence of natural metamorphosis—limits the generalizability of findings to other salamander species. To decipher differences and similarities in the underlying mechanisms of regeneration processes and to better understand evolutionary and ecological influences, comparative studies with other salamander species are essential. Chapter I compares the regenerative abilities of the axolotl with those of the metamorphosing tiger salamander. Larval tiger salamanders exhibit similarly impressive regenerative capacities as axolotls, while post-metamorphic individuals show incomplete fracture healing and delayed regeneration with skeletal anomalies. Chapter II investigates tail regeneration in the tiger salamander during metamorphosis, demonstrating that regeneration continues both during and after this developmental stage, although its speed and structural quality vary. This highlights the impact of metamorphosis on the regeneration process. Chapter III analyzes limb regeneration in six plethodontid salamander species with different lifestyles and habitats. All species exhibit significant regenerative abilities, though anatomical anomalies are common after regeneration. Additionally, correlations between regeneration speed and habitat are identified. Chapter IV examines fin regeneration in lungfish. The observed anomalies resemble those found in salamanders, supporting the hypothesis that epimorphic regenerative abilities have a deep evolutionary origin.