Biology subjects

Sousa, J. F.

Publications and source records attributed to Sousa, J. F..

7 recordsLinked to original sources

bioRxiv2026

A conserved enhancer cluster regulates efnb2 expression in the vertebrate dorsal retina

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.

evolutionary biology↗

bioRxiv2026

Canonical mTOR signaling supports complete fin regeneration

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. Significance StatementThe ability to regenerate complex appendages varies widely among vertebrates, yet the molecular basis of this variation remains poorly understood. Salamanders harbor a uniquely hypersensitive form of mTOR that has been proposed to be essential for limb regeneration. Using Polypterus fin regeneration as a model system, we demonstrate that a canonical vertebrate mTOR protein is compatible with regeneration of a complex, limb-like appendage. Our results show that mTOR acts through selective metabolic, translational, and immune programs. These findings suggest that salamander-specific mTOR hypersensitivity represents a derived enhancement rather than a condition for appendage regeneration and provide insight into the evolutionary foundations of regenerative competence.

developmental biology↗

bioRxiv2025

Evolutionary remodeling of the ventral retina enables aerial vision in the four-eyed fish Anableps anableps

The emergence of evolutionary novelties remains a central challenge in biology, particularly for complex organs such as the vertebrate eye. While visual systems have repeatedly undergone degeneration or loss, examples of innovation are rare and poorly understood. Mechanistic insight into such gains requires tractable systems in which novel visual functions have evolved. Here we leverage the four-eyed fish Anableps anableps to uncover the cellular and molecular basis of adaptation to simultaneous aerial and aquatic light. Combining single-nucleus and spatial transcriptomics, we show that the Anableps ventral retina has been developmentally and functionally reconfigured for aerial vision. In fully aquatic teleosts such as zebrafish, ventral photoreceptors are tuned to red-shifted, downwelling light. In contrast, we show that Anableps exhibits an expanded and diversified ventral cone population encompassing ultraviolet-, blue-, green-, and red-sensitive types, enabling broad-spectrum, high-intensity vision from above water. To estimate Anableps opsin spectral properties, we employed OPTICS, a machine-learning model of opsin spectral tuning, which predicts that the ventrally expressed opn1mw2 opsin is red-shifted relative to typical green-sensitive pigments. Analysis of dorsal-ventral patterning markers reveals an overrepresentation of ventral retinal cell types. Consistent with this, comparative spatial transcriptomic profiling of developing and adult Anableps and the killifish (Fundulus heteroclitus) indicates a dorsal displacement of the dorsal-ventral boundary in Anableps, resulting in a proportionally larger ventral retinal domain. Collectively, our results reveal that Anableps achieved its unique dual visual capacity by developmentally expanding and functionally reprogramming the ventral retina for aerial light detection, an evolutionary innovation that redefines how retinal domains can adapt to radically different visual environments.

evolutionary biology↗

bioRxiv2025

Integrative multi-omics analysis of Polypterus fin regeneration reveals shared and derived fin and limb regeneration mechanisms

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.

evolutionary biology↗

bioRxiv2020

The West African lungfish provides insights into the evolution of tetrapod tail regeneration

Salamanders, frog tadpoles, and diverse lizards have the remarkable ability to regenerate tails. Paleontological data suggests 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 formation of a proliferative blastema and restores original structures, including muscle, skeleton and spinal cord. In contrast to 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 program deployed during lungfish tail regeneration maintains extensive overlap with that of tetrapods, with the upregulation of genes and signaling 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 developmental processes and genetic program 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.

evolutionary biology↗

bioRxiv2019

von Willebrand Factor D and EGF Domains is an evolutionarily conserved and required feature of blastemas capable of multi-tissue 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 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. Further, vwde expression is tightly linked to the ability to regenerate appendages. Functional experiments demonstrate a requirement for vwde in regeneration and indicate that Vwde is a potent mitogen 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.

developmental biology↗

bioRxiv2018

Deep evolutionary origin of limb and fin regeneration

Salamanders and lungfishes are the only sarcopterygians (lobe-finned vertebrates) capable of complete limb and paired fin regeneration, respectively. Among actinopterygians (ray-finned fishes), regeneration after amputation at the fin endoskeleton has only been demonstrated in Polypterid fishes (Cladistia). Whether complete appendage regeneration in sarcopterygians and actinopterygians evolved independently or has a common origin remains unknown. Here we combine fin regeneration assays and comparative RNA-seq analysis to provide support for a common origin of a paired appendage regeneration in osteichthyes (bony vertebrates). We show that, in addition to Polypterids, regeneration after fin endoskeleton amputation occurs in extant representatives of all major actinopterygian clades: the American paddlefish, (Chondrostei), the spotted gar (Holostei), as well as in two cichlid species, the white convict and the oscar (Teleostei). 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 pan-osteichthyes genetic program of appendage regeneration. 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.

evolutionary biology↗