Igor Schneider

The mechanistic target of rapamycin (mTOR) is a conserved regulator of cellular growth, metabolism, and tissue repair. Salamanders possess unique amino acid expansions in mTOR proposed to confer hypersensitive kinase activity, raising the possibility that canonical mTOR is insufficient to support complex appendage regeneration. We tested this hypothesis in the Senegal bichir ( Polypterus senegalus ), a ray-finned fish capable of complete fin regeneration. Fin amputation triggered rapid activation of mTOR signaling, and pharmacological inhibition of mTOR abolished regeneration despite successful wound closure. Single-nucleus RNA sequencing across regenerative stages revealed selective upregulation of mTOR pathway components in proliferating epidermal and connective tissue cells, and in myeloid cells. Inhibition of mTOR specifically attenuated the induction of translational machinery and glycolytic profiles and disproportionately affected myeloid transcriptional states associated with innate immune recognition, interferon-associated signaling, and antigen processing. We propose that salamander-specific mTOR hypersensitivity represents an evolutionary acceleration of an ancestral regenerative program, rather than a prerequisite for limb regeneration.

The vertebrate retina is patterned along its dorsal-ventral (DV) axis by conserved signaling centers, yet the cis -regulatory architecture maintaining these territories remains unclear. Using the four-eyed fish Anableps anableps , which enables physical separation of dorsal and ventral retina, we integrated RNA-seq, ATAC-seq and Hi-C to map DV regulatory landscapes. We identify a 29-kb dorsal-enriched enhancer cluster within a gene poor region between efnb2a and gtpbp8 , embedded in a conserved topologically associating domain spanning efnb2a-gtpbp8-slc10a2 . Dorsal-biased chromatin contacts link this cluster to the efnb2a promoter. The orthologous human region contains a genome-wide significant association with retinal thickness (rs2575134; P = 2e-14). Leveraging the compact genome of Tetraodon nigroviridis , we cloned the orthologous interval and show that it drives dorsal retina-specific reporter expression in zebrafish and mouse. Together, these results identify an evolutionarily conserved regulatory domain controlling retinal axial identity and linking enhancer architecture to human ocular variation. Funding, National Science Foundation.

The mechanistic target of rapamycin (mTOR) pathway is a deeply conserved regulator of cellular growth, metabolism, and tissue repair. In vertebrates, mTOR activity is broadly required for wound healing and regeneration. Recent work in salamanders identified unique amino acid expansions within the mTOR protein. These expansions are thought to confer hypersensitive kinase activity, which was proposed to support rapid translational activation and exceptional limb regeneration capacity. This has raised the possibility that canonical, mammalian-like mTOR signaling may be insufficient to support regeneration of complex appendages. Here, we test this hypothesis by investigating fin regeneration in the Senegal bichir (Polypterus senegalus), a ray-finned fish capable of fully regenerating fins containing bone, cartilage, muscle, and connective tissue with cellular complexity comparable to tetrapod limbs. We show that fin amputation triggers rapid activation of canonical mTOR signaling, and pharmacological inhibition of mTOR with rapamycin abolishes fin regeneration despite successful wound closure. Single-nucleus RNA sequencing (snRNA-seq) across multiple regenerative stages reveals that mTOR pathway components are selectively upregulated in proliferative epidermal and connective tissue cells, and myeloid cell populations. Rather than causing a global shutdown of gene expression, mTOR inhibition specifically dampened the induction of translational machinery and glycolytic programs, two canonical downstream outputs of mTOR signaling. In addition, myeloid cells exhibited pronounced sensitivity to mTOR inhibition, showing attenuated immune-competent and pro-regenerative programs such as interferon-associated signaling, antigen processing, and innate immune coordination. We propose that salamander-specific mTOR hypersensitivity represents an evolutionary acceleration of an ancestral regenerative program, rather than a prerequisite for limb regeneration.

Comparative studies of vertebrate appendages offer a powerful framework for uncovering shared components of an ancestral regeneration toolkit. Here, we employed a multi-omics comparative approach leveraging the regenerative capacity of the axolotl, zebrafish, and Polypterus senegalus , a fish capable of full fin regeneration. We identified conserved markers of proximal and distal blastema territories, shared activation of DNA damage repair, hif1a -mediated hypoxia response, and sequential activation of pro- and anti-inflammatory program. Apical epithelial ridge markers were expressed in both the wound epidermis and distal mesenchyme during limb and fin regeneration. Notably, hif4a -expressing erythrocytes were uniquely associated with proximal limb and fin amputations but not fin rays, while epidermal myoglobin expression was upregulated only in Polypterus and zebrafish fins. Genome-wide chromatin profiling identified candidate regeneration-responsive elements and a conserved enrichment for AP-1 transcription factor binding. Together, these findings identify shared and derived mechanisms of limb and fin regeneration.

Comparative studies of salamander limbs and fish fins may reveal shared components of an ancestral vertebrate appendage regeneration toolkit. However, traditional fish models, such as zebrafish, regenerate only dermal fin rays, which are neither homologous to limbs nor as cellularly complex. To address this, we employed a multi-omics approach leveraging the regenerative capacity of Polypterus senegalus , a fish species capable of full fin regeneration. Single-nucleus RNA-sequencing revealed extensive erythrocyte and myeloid cell infiltration during Polypterus fin regeneration. Spatial transcriptomics identified key cellular contributors to the blastema, while cross-species comparisons with axolotl limbs uncovered both common and divergent expression patterns of wound epidermis and blastema markers. Fin amputation triggered a conserved injury response, including sequential activation of pro- and anti-inflammatory programs and DNA damage repair in cycling cells. Hypoxia responses involved hif1a modulation, consistent with other regenerative models, but uniquely included the expression of hif4a in erythrocytes and the upregulation of a myoglobin gene in the wound epidermis. Furthermore, genome-wide chromatin profiling identified candidate regeneration-responsive elements. Together, these findings establish Polypterus as a powerful comparative model for identifying shared and derived mechanisms of limb and fin regeneration.

The genomes of living lungfishes can inform on the molecular-developmental basis of the Devonian sarcopterygian fish–tetrapod transition. We de novo sequenced the genomes of the African (Protopterus annectens) and South American lungfishes (Lepidosiren paradoxa). The Lepidosiren genome (about 91 Gb, roughly 30 times the human genome) is the largest animal genome sequenced so far and more than twice the size of the Australian (Neoceratodus forsteri)¹ and African² lungfishes owing to enlarged intergenic regions and introns with high repeat content (about 90%). All lungfish genomes continue to expand as some transposable elements (TEs) are still active today. In particular, Lepidosiren’s genome grew extremely fast during the past 100 million years (Myr), adding the equivalent of one human genome every 10 Myr. This massive genome expansion seems to be related to a reduction of PIWI-interacting RNAs and C2H2 zinc-finger and Krüppel-associated box (KRAB)-domain protein genes that suppress TE expansions. Although TE abundance facilitates chromosomal rearrangements, lungfish chromosomes still conservatively reflect the ur-tetrapod karyotype. Neoceratodus’ limb-like fins still resemble those of their extinct relatives and remained phenotypically static for about 100 Myr. We show that the secondary loss of limb-like appendages in the Lepidosiren–Protopterus ancestor was probably due to loss of sonic hedgehog limb-specific enhancers.

For over a century, Lepidosiren paradoxa has been recognized as the single lungfish species in South America. Previous studies have failed to identify morphological differences in lungfish populations inhabiting the Amazon and Parana-Paraguay River basins. Here, we report molecular evidence of cryptic speciation in South American lungfish. Our analysis of mitochondrial gene sequences from 56 L. paradoxa individuals collected in the Amazon and Parana-Paraguay river systems revealed that genetic distances between individuals from different basins were on average 20 times greater than those obtained for individuals that occur in the same basin. Haplotype network did not uncover shared haplotypes between basins. Additionally, the species delimitation analysis showed that the posterior probability for the existence of distinct species is highly significant (PP = 0.99961). The phylogenetic analyses (BI and ML) recovered individuals from distinctive basins in reciprocally monophyletic clades. Our divergence time estimate suggests that the Lepidosiren in the Amazon basin are seven million years split from those that occur in the Parana-Paraguay basin. On the other hand, in agreement with previous reports, our morphological comparison of 20 specimens from the Amazon and 14 specimens from the Parana-Paraguay basins revealed no distinctive morphological features. The combined molecular and morphological results suggest that these two forms may represent cryptic species.