Type 2 vomeronasal receptor expression in the olfactory organ of African lungfish, Protopterus annectens.

The olfactory organ of tetrapods, with few exceptions, comprises the main and accessory organs: olfactory epithelium (OE) and vomeronasal organ (VNO). Unlike tetrapods, teleost fish lack a VNO. However, lungfish, a type of sarcopterygian fish closely related to tetrapods, possesses a lamellar OE similar to the OE of teleosts and a recess epithelium (RecE) resembling the amphibian VNO. The RecE has been hypothesized as a primordial VNO. Olfactory receptors in tetrapods are distinctively expressed in the OE and VNO. For instance, type 2 vomeronasal receptors (V2Rs) in Xenopus are categorized into those exclusively expressed in the OE and those solely expressed in the VNO. It remains unclear whether V2Rs are differentially expressed between the lamellar OE and RecE in lungfish. This study investigated V2R expression in the lamellar OE and RecE of the African lungfish, Protopterus annectens. P. annectens V2Rs were categorized into three groups: those exclusively expressed in the lamellar OE, those exclusively expressed in the RecE, and those expressed in both the lamellar OE and RecE. V2Rs exclusively expressed in the RecE and those expressed in both the lamellar OE and RecE formed a distinct clade in the phylogenetic tree, whereas others were solely expressed in the lamellar OE. These findings suggest that lungfish V2R expression represents an intermediate stage toward complete segregation between V2Rs expressed in the OE and those expressed in the VNO.

Unravelling the mystery of endemic versus translocated populations of the endangered Australian lungfish (Neoceratodus forsteri).

The Australian lungfish is a primitive and endangered representative of the subclass Dipnoi. The distribution of this species is limited to south-east Queensland, with some populations considered endemic and others possibly descending from translocations in the late nineteenth century shortly after European discovery. Attempts to resolve the historical distribution of this species have met with conflicting results based on descriptive genetic studies. Understanding if all populations are endemic or some are the result of, or influenced by, translocation events, has implications for conservation management. In this work, we analysed the genetic variation at three types of markers (mtDNA genomes, 11 STRs and 5196 nuclear SNPs) using the approximate Bayesian computation (ABC) algorithm to compare several demographic models. We postulated different contributions of Mary River and Burnett River gene pools into the Brisbane River and North Pine River populations, related to documented translocation events. We ran the analysis for each marker type separately, and we also estimated the posterior probabilities of the models combining the markers. Nuclear SNPs have the highest power to correctly identify the true model among the simulated datasets (where the model was known), but different marker types typically provided similar answers. The most supported demographic model able to explain the real dataset implies that an endemic gene pool is still present in the Brisbane and North Pine Rivers and coexists with the gene pools derived from past documented translocation events. These results support the view that ABC modelling can be useful to reconstruct complex historical translocation events with contemporary implications, and will inform ongoing conservation efforts for the endangered and iconic Australian lungfish.

Morphology of the cornea and iris in the Australian lungfish Neoceratodus forsteri (Krefft 1870) (Dipnoi): Functional and evolutionary perspectives of transitioning from an aquatic to a terrestrial environment.

The Australian lungfish, Neoceratodus forsteri (Krefft 1870), is the sole extant member of the Ceratodontidae within the Dipnoi, a small order of sarcopterygian (lobe-finned) fishes, that is thought to be the earliest branching species of extant lungfishes, having changed little over the last 100 million years. To extend studies on anatomical adaptations associated with the fish-tetrapod transition, the ultrastructure of the cornea and iris is investigated using light and electron (transmission and scanning) microscopy to investigate structure-function relationships and compare these to other vertebrate corneas (other fishes and tetrapods). In contrast to previous studies, the cornea is found to have only three main components, comprising an epithelium with its basement membrane, a stroma with a Bowman's layer and an endothelium, and is not split into a dermal (secondary) spectacle and a scleral cornea. The epithelial cells are large, relatively low in density and similar to many species of non-aquatic tetrapods and uniquely possess numerous surface canals that contain and release mucous granules onto the corneal surface to avoid desiccation. A Bowman's layer is present and, in association with extensive branching and anastomosing of the collagen fibrils, may be an adaptation for the inhibition of swelling and/or splitting of the stroma during its amphibious lifestyle. The dorsal region of the stroma possesses aggregations of pigment granules that act as a yellow, short wavelength-absorbing filter during bright light conditions. Desçemet's membrane is absent and replaced by an incomplete basement membrane overlying a monocellular endothelium. The iris is pigmented, well-developed, vascularised and contractile containing reflective crystals anteriorly. Based upon its ultrastructure and functional adaptations, the cornea of N. forsteri is more similar to amphibians than to other bony fishes and is well-adapted for an amphibious lifestyle.

Unveiling the expansion of keratin genes in lungfishes: a possible link to terrestrial adaptation.

Keratins are intermediate filament proteins that are important for epidermal strength and protection from desiccation. Keratin genes are highly duplicated and have diversified by forming two major clusters in the genomes of terrestrial vertebrates. The keratin genes of lungfishes, the closest fish to tetrapods, have not been studied at the genomic level, despite the importance of lungfishes in terrestrial adaptation. Here, we identified keratin genes in the genomes of two lungfish species and performed syntenic and phylogenetic analyses. Additionally, we identified keratin genes from two gobies and two mudskippers, inhabiting underwater and terrestrial environments. We found that in lungfishes, keratin genes were duplicated and diversified within two major clusters, similar to but independent of terrestrial vertebrates. By contrast, keratin genes were not notably duplicated in mudskippers. The results indicate that keratin gene duplication occurred repeatedly in lineages close to tetrapods, but not in teleost fish, even in species adapted to terrestrial environments.

Relaxation of Natural Selection in the Evolution of the Giant Lungfish Genomes.

Nonadaptive hypotheses on the evolution of eukaryotic genome size predict an expansion when the process of purifying selection becomes weak. Accordingly, species with huge genomes, such as lungfish, are expected to show a genome-wide relaxation signature of selection compared with other organisms. However, few studies have empirically tested this prediction using genomic data in a comparative framework. Here, we show that 1) the newly assembled transcriptome of the Australian lungfish, Neoceratodus forsteri, is characterized by an excess of pervasive transcription, or transcriptional leakage, possibly due to suboptimal transcriptional control, and 2) a significant relaxation signature in coding genes in lungfish species compared with other vertebrates. Based on these observations, we propose that the largest known animal genomes evolved in a nearly neutral scenario where genome expansion is less efficiently constrained.

Functional characterization of melanocortin 2 receptor (Mc2r) from a lobe-finned fish (Protopterus annectens) and insights into the molecular evolution of melanocortin receptors.

Recent studies from our group on melanocortin 2 receptors (Mc2r) from basal families of actinopterygians have served to resolve that Mrap1 dependence and ACTH selectivity are features of even the most basal ray-finned fishes. However, there have been no studies on Mc2r function of the basal sarcopterygians, the lobe-finned fishes, represented by the extant members coelacanths and lungfishes. Here, we offer the first molecular and functional characterization of an Mc2r from a lobe-finned fish, the West African lungfish (Protopterus annectens). Plasmids containing cDNA constructs of lungfish (lf) Mc2r and Mrap1 were expressed in mammalian and zebrafish cell lines. Cells were then stimulated by human ACTH(1-24) and melanocyte stimulating hormone (α-MSH), as well as alanine-substituted analogs of hACTH(1-24) targeting residues within the H6F7R8W9 and K15K16R17R18P19 motifs. Activation of lfMc2r was assessed using a cAMP-responsive luciferase reporter gene assay. In these assays, lfMc2r required co-expression with lfMrap1, was selective for ACTH over α-MSH at physiological concentrations of the ligands, and was completely inhibited by multiple-alanine substitutions of the HFRW (A6-9) and KKRRP (A15-19) motifs. Single- and partial-alanine substitutions of the HFRW and KKRRP motifs varied in their impacts on receptor-ligand affinity from having no effect to completely inhibiting lfMc2r activation. This characterization of the Mc2r of a lobe-finned fish fulfills the last major extant vertebrate group for which Mc2r function had yet to be characterized. In doing so, we resolve that all basal bony vertebrate groups exhibit Mc2r function that substantially differs from that of the cartilaginous fishes, indicating that rapid and dramatic shift in Mc2r function occurred between the radiation of cartilaginous fishes and the emergence of bony fishes. We support this interpretation with a molecular clock analysis of the melanocortin receptors, which demonstrates the uniquely high rate of sequence divergence in Mc2r. Much remains to be understood regarding the molecular evolution of Mc2r during the early radiation of vertebrates that resulted in the derived functional characteristics of Mrap1 dependence and exclusive selectivity for ACTH.

Immunolocalization of Some Epidermal Proteins and Glycoproteins in the Growing Skin of the Australian Lungfish (Neoceratodus forsteri).

Here we report the immunolocalization of mucin, nestin, elastin and three glycoproteins involved in tissue mineralization in small and large juveniles of Neoceratodus forsteri. Both small and larger juvenile epidermis are mucogenic and contain a diffuse immunolabeling for nestin. Sparse PCNA-labeled cells, indicating proliferation, are found in basal and suprabasal epidermal layers. No scales are formed in small juveniles but are present in a 5 cm long juvenile and in larger juveniles. Elastin and a mineralizing matrix are localized underneath the basement membrane of the tail epidermis where lepidotriches are forming. The latter appears as "circular bodies" in cross sections and are made of elongated cells surrounding a central amorphous area containing collagen and elastin-like proteins that undergo calcification as evidenced using the von Kossa staining. However, the first calcification sites are the coniform teeth of the small juveniles of 2-3 cm in length. In the superficial dermis of juveniles (16-26 cm in length) where scales are formed, the spinulated outer bony layer (squamulin) of the elasmoid scales contains osteonectin, alkaline phosphatase, osteopontin, and calcium deposits that are instead absent in the underlying layer of elasmodin. In particular, these glycoproteins are localized along the scale margin in juveniles where scales grow, as indicated by the presence of PCNA-labeled cells (proliferating). These observations suggest a continuous deposition of new bone during the growth of the scales, possibly under the action of these mineralizing glycoproteins, like in the endoskeleton of terrestrial vertebrates.

Type 1 vomeronasal receptor expression in juvenile and adult lungfish olfactory organ.

Lungfish are the most closely related fish to tetrapods. The olfactory organ of lungfish contains lamellae and abundant recesses at the base of lamellae. Based on the ultrastructural and histochemical characteristics, the lamellar olfactory epithelium (OE), covering the surface of lamellae, and the recess epithelium, contained in the recesses, are thought to correspond to the OE of teleosts and the vomeronasal organ (VNO) of tetrapods. With increasing body size, the recesses increase in number and distribution range in the olfactory organ. In tetrapods, the expression of olfactory receptors is different between the OE and VNO; for instance, the type 1 vomeronasal receptor (V1R) is expressed only in the OE in amphibians and mainly in the VNO in mammals. We recently reported that V1R-expressing cells are contained mainly in the lamellar OE but also rarely in the recess epithelium in the olfactory organ of lungfish of approximately 30 cm body length. However, it is unclear whether the distribution of V1R-expressing cells in the olfactory organ varies during development. In this study, we compared the expression of V1Rs in the olfactory organs between juveniles and adults of the African lungfish Protopterus aethiopicus and South American lungfish, Lepidosiren paradoxa. The density of V1R-expressing cells was higher in the lamellae than in the recesses in all specimens evaluated, and this pattern was more pronounced in juveniles than adults. In addition, the juveniles showed a higher density of V1R-expressing cells in the lamellae compared with the adults. Our results imply that differences in lifestyle between juveniles and adults are related to differences in the density of V1R-expressing cells in the lamellae of lungfish.

Morpho-functional changes of lungfish Protopterus dolloi skin in the shift from freshwater to aestivating conditions.

African dipnoi (Protopterus sp.) are obligate air-breathing fish that, during dry season, may experience a period of dormancy named aestivation. Aestivation is characterized by complete reliance on pulmonary breathing, general decrease of metabolism and down-regulation of respiratory and cardiovascular functions. To date, little is known about morpho-functional rearrangements induced by aestivation in the skin of African lungfishes. Our study aims to identify, in the skin of P. dolloi, structural modifications and stress-induced molecules in response to short-term (6 days) and long-term (40 days) aestivation. Light microscopy showed that short-term aestivation induces major reorganization, with narrowing of epidermal layers and decrease of mucous cells; prolonged aestivation is characterized by regenerative processes and re-thickening of epidermal layers. Immunofluorescence reveals that aestivation correlates with an increased oxidative stress and changes of Heat Shock Proteins expression, suggesting a protective role for these chaperons. Our findings revealed that lungfish skin undergoes remarkable morphological and biochemical readjustments in response to stressful conditions associated with aestivation.

Expression of SATB1 and SATB2 in the brain of bony fishes: what fish reveal about evolution.

Satb1 and Satb2 belong to a family of homeodomain proteins with highly conserved functional and regulatory mechanisms and posttranslational modifications in evolution. However, although their distribution in the mouse brain has been analyzed, few data exist in other non-mammalian vertebrates. In the present study, we have analyzed in detail the sequence of SATB1 and SATB2 proteins and the immunolocalization of both, in combination with additional neuronal markers of highly conserved populations, in the brain of adult specimens of different bony fish models at key evolutionary points of vertebrate diversification, in particular including representative species of sarcopterygian and actinopterygian fishes. We observed a striking absence of both proteins in the pallial region of actinopterygians, only detected in lungfish, the only sarcopterygian fish. In the subpallium, including the amygdaloid complex, or comparable structures, we identified that the detected expressions of SATB1 and SATB2 have similar topologies in the studied models. In the caudal telencephalon, all models showed significant expression of SATB1 and SATB2 in the preoptic area, including the acroterminal domain of this region, where the cells were also dopaminergic. In the alar hypothalamus, all models showed SATB2 but not SATB1 in the subparaventricular area, whereas in the basal hypothalamus the cladistian species and the lungfish presented a SATB1 immunoreactive population in the tuberal hypothalamus, also labeled with SATB2 in the latter and colocalizing with the gen Orthopedia. In the diencephalon, all models, except the teleost fish, showed SATB1 in the prethalamus, thalamus and pretectum, whereas only lungfish showed also SATB2 in prethalamus and thalamus. At the midbrain level of actinopterygian fish, the optic tectum, the torus semicircularis and the tegmentum harbored populations of SATB1 cells, whereas lungfish housed SATB2 only in the torus and tegmentum. Similarly, the SATB1 expression in the rhombencephalic central gray and reticular formation was a common feature. The presence of SATB1 in the solitary tract nucleus is a peculiar feature only observed in non-teleost actinopterygian fishes. At these levels, none of the detected populations were catecholaminergic or serotonergic. In conclusion, the protein sequence analysis revealed a high degree of conservation of both proteins, especially in the functional domains, whereas the neuroanatomical pattern of SATB1 and SATB2 revealed significant differences between sarcopterygians and actinopterygians, and these divergences may be related to the different functional involvement of both in the acquisition of various neural phenotypes.

Aestivation induces widespread transcriptional changes in the African lungfish.

Aestivation is a special ability possessed by some animals to cope with hot and dry environments utilizing dormancy. At a macroscopic level, dormant animals stop moving and eating. At the microscopic level, the expression of a large number of genes in these animals is strictly controlled. However, little is known about what changes occur during aestivation, especially in fish. In this study, we used transcriptome analysis to examine what changes occur in the gills and lungs of the African lungfish (Protopterus annectens) during the maintenance phase of aestivation and speculated on their causes. We found that aestivating transcriptomes were highly similar between gills and lungs. We also found that some genes showed differential expression or alternative splicing, which may be associated with different organs. In addition, differential expression analysis revealed that the lungs maintained significantly higher bioactivity during aestivation, which suggests that the main respiratory organ in aestivating lungfish can transform. Our study provides a reference point for studying the relationship between aestivation and hibernation and further increases understanding of aestivation.

Type 1 vomeronasal receptors expressed in the olfactory organs of two African lungfish, Protopterus annectens and Protopterus amphibius.

Lungfish are the fish related most closely to tetrapods. The olfactory organ of lungfish contains two distinct sensory epithelia: the lamellar olfactory epithelium (OE) and the recess epithelium (RecE). Based on their ultrastructural and histological characteristics, the lamellar OE and the RecE are considered to correspond respectively to the teleost OE and a primitive vomeronasal organ (VNO). In tetrapods, the OE and VNO have been shown to express different families of olfactory receptors; for example, in mammals, the OE expresses odorant receptors and trace amine-associated receptors, while the VNO expresses type 1 (V1Rs) and type 2 (V2Rs) vomeronasal receptors. In the present study, we examined the expression of V1Rs in the olfactory organs of two African lungfish, Protopterus annectens and Protopterus amphibius. RNA sequencing and phylogenetic analyses identified 29 V1R genes in P. annectens and 50 V1R genes in P. amphibius. Most V1Rs identified in these lungfish were classified as the tetrapod-type V1Rs initially found in tetrapods and distinct from fish-type V1Rs. In teleost, which all lack a VNO, all olfactory receptors are expressed in the OE, while in Xenopus V1Rs are expressed exclusively in the OE, and not in the VNO. In situ hybridization analysis indicated that lungfish V1Rs were expressed mainly in the lamellar OE and rarely in the RecE. These results imply that V1R expression in lungfish represents an intermediate step toward the complete segregation of V1R expression between the OE and VNO, reflecting the phylogenetic position of lungfish between teleosts and amphibians.

How the evolution of air breathing shaped hippocampal function.

To make maps from airborne odours requires dynamic respiratory patterns. I propose that this constraint explains the modulation of memory by nasal respiration in mammals, including murine rodents (e.g. laboratory mouse, laboratory rat) and humans. My prior theories of limbic system evolution offer a framework to understand why this occurs. The answer begins with the evolution of nasal respiration in Devonian lobe-finned fishes. This evolutionary innovation led to adaptive radiations in chemosensory systems, including the emergence of the vomeronasal system and a specialization of the main olfactory system for spatial orientation. As mammals continued to radiate into environments hostile to spatial olfaction (air, water), there was a loss of hippocampal structure and function in lineages that evolved sensory modalities adapted to these new environments. Hence the independent evolution of echolocation in bats and toothed whales was accompanied by a loss of hippocampal structure (whales) and an absence of hippocampal theta oscillations during navigation (bats). In conclusion, models of hippocampal function that are divorced from considerations of ecology and evolution fall short of explaining hippocampal diversity across mammals and even hippocampal function in humans. This article is part of the theme issue 'Systems neuroscience through the lens of evolutionary theory'.

Aldosterone and dexamethasone activate African lungfish mineralocorticoid receptor: Increased activation after removal of the amino-terminal domain.

Aldosterone, the main physiological mineralocorticoid in humans and other terrestrial vertebrates, first appears in lungfish, which are lobe-finned fish that are forerunners of terrestrial vertebrates. Aldosterone activation of the MR regulates internal homeostasis of water, sodium and potassium, which was critical in the conquest of land by vertebrates. We studied transcriptional activation of the slender African lungfish MR by aldosterone, other corticosteroids and progesterone and find that aldosterone, 11-deoxycorticosterone, 11-deoxycortisol and progesterone have half-maximal responses (EC50 s) below 1 nM and are potential physiological mineralocorticoids. In contrast, EC50 s for corticosterone and cortisol were 23 nM and 66 nM, respectively. Unexpectedly, truncated lungfish MR, consisting of the DNA-binding, hinge and steroid-binding domains, had a stronger response to corticosteroids and progesterone than full-length lungfish MR, indicating that the N-terminal domain represses steroid activation of lungfish MR, unlike human MR in which the N-terminal domain contains an activation function. BLAST searches of GenBank did not retrieve a GR ortholog, leading us to test dexamethasone and triamcinolone for activation of lungfish MR. At 10 nM, both synthetic glucocorticoids are about 4-fold stronger than 10 nM aldosterone in activating full-length lungfish MR, leading us to propose that lungfish MR also functions as a GR.

Earliest migratory cephalic NC cells are potent to differentiate into dental ectomesenchyme of the two lungfish dentitions: tetrapodomorph ancestral condition of unconstrained capability of mesencephalic NC cells to form oral teeth.

Reciprocal interactions between epithelial and neural crest-derived mesenchymal cells have been recognized in the evolutionary modulation of tetrapod odontodes, skeletal structures that include the teeth and tooth-integrated basal tissue. Using cell-tracking experiments, it has been demonstrated that mandibular neural crest cells, labelled during migration, extensively populate dental papillae of all tooth phenotypes of the lobe-finned fish, the Australian lungfish (Neoceratodus forsteri). Here, I report on an extension of this experimental study that earliest migrating NC cells are able to differentiate into odontogenic ectomesenchyme. Using vital dye cell-tracking to mark the mesencephalic neural crest prior to migration, I have found that the corresponding population of earliest migratory cells selectively relocated to dental papillae of both temporary and permanent dentitions of Neoceratodus. I noticed a gradient in distribution of the labelled cells which populated posterior teeth, pterygoid and prearticular (including associated trabecular and Meckelian cartilages; major relocation) much more densely than those in anterior marginal positions, temporary and vomeral permanent teeth (minor relocation). Contrary to mice and zebrafish, the odontogenic potency of mesencephalic neural crest cells is already programmed at the onset of the migration event in lungfish. This may imply that the morphogenic potential of mesencephalic neural crest cells to form teeth has been heterochronically shifted and constrained to later migratory populations of neural crest cells during the developmental evolution of derived tetrapods, or/and arrested in their expression in the oral development of some modern osteichthyans.

Morphology and histology of the olfactory organ of two African lungfishes, Protopterus amphibius and P. dolloi (Lepidosirenidae, Dipnoi).

The olfactory organs of two African lungfishes, Protopterus amphibius and P. dolloi, were investigated using a stereo microscope and a compound light microscope and were described anatomically, histologically, and histochemically. Like other lungfishes, these species present the following general features: i) elongated olfactory chamber (OC), ii) anterior nostril at the ventral tip of the upper lip, iii) posterior nostril on the palate of the oral cavity, iv) lamellae with multiple cell types such as olfactory receptor neurons, supporting cells, basal cells, lymphatic cells, and mucous cells (MC), and vi) vomero-like epithelial crypt (VEC) made of glandular epithelium (GE) and crypt sensory epithelium. Some of these features exhibit differences between species: MCs are abundant in both the lamellar and inner walls of the OC in P. amphibius but occur only in lamellae in P. dolloi. On the other hand, some between feature differences are consistent across species: the GE of both P. amphibius and P. dolloi is strongly positive for Alcian blue (pH 2.5)-periodic acid Schiff (deep violet coloration), and positive with hematoxylin and eosin and with Masson's trichrome (reddish-brown staining), unlike the MCs of the two species which stain dark red with both Alcian blue (pH 2.5)-periodic acid Schiff and Masson's trichrome but respond faintly to hematoxylin and eosin. The differing abundance of MCs in the two lungfishes might reflect different degrees in aerial exposure of the olfactory organ, while the neutral and acid mucopolysaccharide-containing VEC, as indicated by staining properties of the MCs, is evolutionary evidence that P. amphibius and P. dolloi are the closest living relatives to tetrapods, at least in the order Dipnoi.

Pseudocapillaria (Ichthyocapillaria) bumpi n. sp. (Nematoda: Capillariidae) Parasitising West African Lungfish Protopterus annectens (Owen, 1839) (Lepidosireniformes: Protopteridae) in Mozambique and Its Phylogenetic Position Within Capillariid Nematodes.

INTRODUCTION: Pseudocapillaria (Ichthyocapillaria) bumpi n. sp. (Nematoda: Capillariidae) is described from specimens infecting the digestive tract of the West African lungfish, Protopterus annectens (Owen) (Lepidosireniformes: Protopteridae). METHODS: Nematodes were collected in Karingani Game Reserve, Southern Mozambique and studied using light microscopy; obtained sequences of the 18S ribosomal (18S rRNA) gene used for phylogenetic studies. RESULTS: The new species is assigned to Pseudocapillaria Freitas, 1959 by having a stichosome consisting of a single row of stichocytes and ventrolateral lobes on the male tail as well as by lacking spines on the specular sheath, caudal alae, a membranous caudal bursa, and a lateral expansion of the caudal end. It is also assigned to the sub-genus Ichthyocapillaria Moravec, 1982 by having a membrane between the ventrolateral lobes and by lacking a vulvar appendage. Pseudocapillaria (I.) bumpi n. sp. differs from its nominal congeners by having a comparatively large body size with relatively long spicule, bearing the thin membrane non-extending further than ventrolateral lobes in males and relatively smaller eggs without protruding polar plugs. It is the 20th species of the genus, the first from the Afrotropical Realm, and the first from any lungfish species. Phylogenetic analyses suggested that P. (I.) bumpi is deeply nested in a clade that associates species of the genera Pearsonema, Aonchotheca and Baruscapillaria.

Mandibular musculature constrains brain-endocast disparity between sarcopterygians.

The transition from water to land by the earliest tetrapods in the Devonian Period is seen as one of the greatest steps in evolution. However, little is understood concerning changes in brain morphology over this transition. Here, we determine the brain-braincase relationship in fishes and basal lissamphibians as a proxy to elucidate the changes that occurred over the fish-tetrapod transition. We investigate six basal extant sarcopterygians spanning coelacanths to salamanders (Latimeria chalumnae, Neoceratodus, Protopterus aethiopicus, P. dolloi, Cynops, Ambystoma mexicanum) using micro-CT and MRI and quantify the brain-braincase relationship in these extant taxa. Our results show that regions of lowest brain-endocast disparity are associated with regions of bony reinforcement directly adjacent to masticatory musculature for the mandible except in Neoceratodus and Latimeria. In Latimeria this deviation from the trend can be accounted for by the possession of an intracranial joint and basicranial muscles, whereas in Neoceratodus difference is attributed to dermal bones contributing to the overall neurocranial reinforcement. Besides Neoceratodus and Latimeria, regions of low brain-endocast disparity occur where there is less reinforcement away from high mandibular muscle mass, where the trigeminal nerve complex exits the braincase and where endolymphatic sacs occupy space between the brain and braincase wall. Despite basal tetrapods possessing reduced adductor muscle mass and a different biting mechanism to piscine sarcopterygians, regions of the neurocranium lacking osteological reinforcement in the basal tetrapods Lethiscus and Brachydectes broadly correspond to regions of high brain-endocast disparity seen in extant taxa.

Salamander-like tail regeneration in the West African lungfish.

Salamanders, frog tadpoles and diverse lizards have the remarkable ability to regenerate tails. Palaeontological data suggest that this capacity is plesiomorphic, yet when the developmental and genetic architecture of tail regeneration arose is poorly understood. Here, we show morphological and molecular hallmarks of tetrapod tail regeneration in the West African lungfish Protopterus annectens, a living representative of the sister group of tetrapods. As in salamanders, lungfish tail regeneration occurs via the formation of a proliferative blastema and restores original structures, including muscle, skeleton and spinal cord. In contrast with lizards and similar to salamanders and frogs, lungfish regenerate spinal cord neurons and reconstitute dorsoventral patterning of the tail. Similar to salamander and frog tadpoles, Shh is required for lungfish tail regeneration. Through RNA-seq analysis of uninjured and regenerating tail blastema, we show that the genetic programme deployed during lungfish tail regeneration maintains extensive overlap with that of tetrapods, with the upregulation of genes and signalling pathways previously implicated in amphibian and lizard tail regeneration. Furthermore, the lungfish tail blastema showed marked upregulation of genes encoding post-transcriptional RNA processing components and transposon-derived genes. Our results show that the developmental processes and genetic programme of tetrapod tail regeneration were present at least near the base of the sarcopterygian clade and establish the lungfish as a valuable research system for regenerative biology.

von Willebrand factor D and EGF domains is an evolutionarily conserved and required feature of blastemas capable of multitissue appendage regeneration.

Regenerative ability varies tremendously across species. A common feature of regeneration of appendages such as limbs, fins, antlers, and tails is the formation of a blastema-a transient structure that houses a pool of progenitor cells that can regenerate the missing tissue. We have identified the expression of von Willebrand factor D and EGF domains (vwde) as a common feature of blastemas capable of regenerating limbs and fins in a variety of highly regenerative species, including axolotl (Ambystoma mexicanum), lungfish (Lepidosiren paradoxa), and Polpyterus (Polypterus senegalus). Further, vwde expression is tightly linked to the ability to regenerate appendages in Xenopus laevis. Functional experiments demonstrate a requirement for vwde in regeneration and indicate that Vwde is a potent growth factor in the blastema. These data identify a key role for vwde in regenerating blastemas and underscore the power of an evolutionarily informed approach for identifying conserved genetic components of regeneration.