Deep evolutionary origin of limb and fin regeneration.

Salamanders and lungfishes are the only sarcopterygians (lobe-finned vertebrates) capable of paired appendage regeneration, regardless of the amputation level. Among actinopterygians (ray-finned fishes), regeneration after amputation at the fin endoskeleton has only been demonstrated in polypterid fishes (Cladistia). Whether this ability evolved independently in sarcopterygians and actinopterygians or has a common origin remains unknown. Here we combine fin regeneration assays and comparative RNA-sequencing (RNA-seq) analysis of Polypterus and axolotl blastemas to provide support for a common origin of paired appendage regeneration in Osteichthyes (bony vertebrates). We show that, in addition to polypterids, regeneration after fin endoskeleton amputation occurs in extant representatives of 2 other nonteleost actinopterygians: the American paddlefish (Chondrostei) and the spotted gar (Holostei). Furthermore, we assessed regeneration in 4 teleost species and show that, with the exception of the blue gourami (Anabantidae), 3 species were capable of regenerating fins after endoskeleton amputation: the white convict and the oscar (Cichlidae), and the goldfish (Cyprinidae). Our comparative RNA-seq analysis of regenerating blastemas of axolotl and Polypterus reveals the activation of common genetic pathways and expression profiles, consistent with a shared genetic program of appendage regeneration. Comparison of RNA-seq data from early Polypterus blastema to single-cell RNA-seq data from axolotl limb bud and limb regeneration stages shows that Polypterus and axolotl share a regeneration-specific genetic program. Collectively, our findings support a deep evolutionary origin of paired appendage regeneration in Osteichthyes and provide an evolutionary framework for studies on the genetic basis of appendage regeneration.

Eye development in the four-eyed fish Anableps anableps: cranial and retinal adaptations to simultaneous aerial and aquatic vision.

The unique eyes of the four-eyed fish Anableps anableps have long intrigued biologists. Key features associated with the bulging eye of Anableps include the expanded frontal bone and the duplicated pupils and cornea. Furthermore, the Anableps retina expresses different photoreceptor genes in dorsal and ventral regions, potentially associated with distinct aerial and aquatic stimuli. To gain insight into the developmental basis of the Anableps unique eye, we examined neurocranium and eye ontogeny, as well as photoreceptor gene expression during larval stages. First, we described six larval stages during which duplication of eye structures occurs. Our osteological analysis of neurocranium ontogeny revealed another distinctive Anablepid feature: an ossified interorbital septum partially separating the orbital cavities. Furthermore, we identified the onset of differences in cell proliferation and cell layer density between dorsal and ventral regions of the retina. Finally, we show that differential photoreceptor gene expression in the retina initiates during development, suggesting that it is inherited and not environmentally determined. In sum, our results shed light on the ontogenetic steps leading to the highly derived Anableps eye.

Morphological And Molecular Analyses of an Anatomical Novelty: The Pelvic Fin Filaments of the South American Lungfish.

The pelvic fins of male South American lungfish, Lepidosiren paradoxa, are adorned with a distinctive array of filaments, which grow and become highly vascularized during the breeding season. The resemblance between these pelvic fin filaments (PFFs) and external gills of other vertebrates suggested that this gill-like structure was used for physiological gas exchange. It has been proposed that the unique pelvic fin of male L. paradoxa is used for release of oxygen from its blood into the environment in order to aerate its nesting brood, or, conversely, as an auxiliary respiratory organ by absorbing oxygen from the environment into its bloodstream. Here, we employed histology, scanning electron microscopy (SEM) and quantitative PCR (qPCR) to assess whether the morphology and molecular profile of PFFs are compatible with a role in gas exchange. First, we closely examined its external morphology and showed that PFFs develop from short papillae during the rainy season, but remain covered by a thick nonvascularized epithelium. Histological examination confirmed that capillaries within the filaments are separated from the exterior by a basement membrane and a stratified epithelium composed of four to five cell layers. In addition, SEM analysis revealed significant differences between the fin filament epithelium and typical gill epithelium. Finally, our qPCR results showed that five genes commonly expressed in gills were downregulated in PFFs relative to their expression in regular pectoral fin epidermis. Collectively, our results do not directly support a role for PFFs, commonly referred to as "limb gills", in oxygen release or uptake.

Tetrapod limb and sarcopterygian fin regeneration share a core genetic programme.

Salamanders are the only living tetrapods capable of fully regenerating limbs. The discovery of salamander lineage-specific genes (LSGs) expressed during limb regeneration suggests that this capacity is a salamander novelty. Conversely, recent paleontological evidence supports a deeper evolutionary origin, before the occurrence of salamanders in the fossil record. Here we show that lungfishes, the sister group of tetrapods, regenerate their fins through morphological steps equivalent to those seen in salamanders. Lungfish de novo transcriptome assembly and differential gene expression analysis reveal notable parallels between lungfish and salamander appendage regeneration, including strong downregulation of muscle proteins and upregulation of oncogenes, developmental genes and lungfish LSGs. MARCKS-like protein (MLP), recently discovered as a regeneration-initiating molecule in salamander, is likewise upregulated during early stages of lungfish fin regeneration. Taken together, our results lend strong support for the hypothesis that tetrapods inherited a bona fide limb regeneration programme concomitant with the fin-to-limb transition.