Evolutionary Study Uncovers Ancient Genetic Switch Governing Vertebrate Muscle Development

A comparative genomic investigation has mapped the deep evolutionary architecture of an ancient cis-regulatory switch governing the Myostatin gene across vertebrates, tracing its origins back to the common ancestor of cartilaginous and bony lineages over 450 million years ago. Published in Developmental Biology Advances by an international team led by Carolina Stefano Mantovani and colleagues, the study resolved how the Myostatin promoter and enhancer evolved through ancestral duplications, transcription factor binding site modifications, and lineage-specific diversifications. By analyzing regulatory sequences spanning jawless, cartilaginous, and bony vertebrates alongside embryonic gene expression assays in avian and mammalian models, the investigators uncovered how structural rearrangements in non-coding DNA altered transcriptional control over skeletal muscle development. Their findings demonstrate that an ancient core promoter underwent tandem duplication early in osteichthyan history, establishing a flexible bipartite genomic platform that subsequently accumulated specialized regulatory motifs associated with anatomical diversification, metabolic adaptations, and transitions in vertebrate locomotion.

The Myostatin gene, designated MSTN, encodes a secreted transforming growth factor-beta superfamily signaling protein that functions as an essential negative regulator of skeletal muscle growth across animal species. During embryonic development, Myostatin balances the proliferation and differentiation of myogenic precursor cells before these progenitors generate multinucleated skeletal muscle fibers. It modulates intracellular signaling by regulating the cyclin-dependent kinase inhibitor p21 alongside key myogenic factors such as MYOD, thereby enforcing cell cycle exit and initiating differentiation. Disruptions in the Myostatin coding sequence produce striking hypermuscular phenotypes documented in knockout mice, naturally occurring mammalian double-muscled mutants, and agricultural livestock. Furthermore, single-nucleotide polymorphisms within the gene correlate with variations in body weight and muscularity in poultry and commercial teleost fish. Nevertheless, while coding mutations are well cataloged, the evolutionary trajectory and structural variation of Myostatin non-coding cis-regulatory elements have remained largely uncharacterized across vertebrate taxa.

To reconstruct this evolutionary history, the investigators implemented a phylogenetic framework utilizing genomic data from major public repositories, including the National Center for Biotechnology Information and Ensembl databases. The team surveyed representative species from primary vertebrate clades, including agnathans like lampreys and hagfish, chondrichthyans comprising sharks, rays, and chimaeras, actinopterygians including holosteans and teleosts, and sarcopterygians spanning coelacanths, amphibians, sauropsids, and mammals. Distant homology BLASTN searches utilized human and chicken promoter-enhancer sequences alongside the ortholog from spotted gar, Lepisosteus oculatus, an ancient holostean fish whose slow-evolving genome lacks teleost whole-genome duplications. For amphibians, the mstn1 regulatory region of Xenopus tropicalis served as an analytical query due to elevated sequence identity compared to the paralogous mstn2 locus. Retrieved sequences were aligned and curated using BioEdit and ClustalW in MEGA software, enabling calculation of intraclade sequence identities and coefficients of variation across standardized promoter alignments.

Comparative sequence alignments revealed that functional Myostatin promoter and enhancer orthologs are absent from extant jawless agnathans, including lamprey and hagfish genomes, and could not be detected in the elephant shark, Callorhinchus milii. However, homologous regulatory sequences were identified across all other examined jawed vertebrates, demonstrating that this regulatory switch arose in ancestral gnathostomes prior to the divergence of cartilaginous and bony lineages. Multiple sequence alignments delineated a conserved core promoter shared across jawed vertebrates containing basal regulatory elements, including a canonical TATA box, an NF-Y recognition sequence, and a MEIS1 transcription factor binding motif. Intraclade variation metrics showed that cartilaginous fishes maintained exceptionally high conservation across the entire promoter-enhancer region. Conversely, bony vertebrates exhibited marked evolutionary dynamism: teleost fishes demonstrated such pronounced divergence that only their core promoter could be aligned, whereas birds displayed significantly higher intraclade regulatory sequence identity than mammalian lineages.

Bioinformatic scanning utilizing the MultiTF algorithm and TRANSFAC motif libraries uncovered systematic gains, losses, and reshuffling of transcription factor binding sites across bony vertebrate lineages. The analysis established that while MEIS1, NF-Y, and the TATA box were ancestral gnathostome features, a CREB binding site became integrated into the core promoter during the early evolution of bony vertebrates. Subsequent evolutionary transitions altered this regulatory repertoire; teleost fishes and amphibians lost the conserved core MEIS1 motif, leaving amphibians with a minimal promoter containing only TATA box and NF-Y sites. In contrast, amniotes acquired novel PAX8 and FXR binding motifs situated upstream from the conserved core, increasing regulatory complexity. Within specific amniote lineages, additional alterations emerged: squamate reptiles lost conserved PAX8 and FXR sites, while Neoaves lost FXR and MEIS1 motifs. Among mammals, Afrotherian species exhibited diverse site losses, vespertilionid bats lacked both FXR and PAX8 elements, and the Bovidae family experienced disruption of the ancestral CREB binding site.

To explain how promoter functionality persisted despite widespread lineage-specific deletion of canonical motifs, the authors evaluated transcription factor binding site turnover across divergent vertebrate genomes. Turnover describes an evolutionary process where homologous regulatory activity is conserved despite sequence divergence through the emergence of compensatory binding motifs at alternative positions within the regulatory locus. The research team identified striking evidence of functional compensation in the cape golden mole, Chrysochloris asiatica, which possesses a disrupted ancestral TATA box but acquired a de novo functional TATA motif elsewhere in its promoter. Similar turnover dynamics generated reiterated core motifs across multiple clades, including duplicate TATA boxes in numerous sauropsid and mammalian genomes. Furthermore, a third functional TATA box was detected in Xenopus tropicalis, the cape elephant shrew Elephantulus edwardii, domestic horses, and donkeys. Widespread gains and positional relocations of CREB, NF-Y, and MEIS1 sites demonstrate that non-coding divergence frequently preserves regulatory control through flexible compensatory motif rearrangements.

A major structural insight reported in the study is the discovery of an ancient bipartite organization within the amniote Myostatin promoter and enhancer, arising from an ancestral tandem duplication of the core promoter element. Comparative alignment against the slow-evolving spotted gar genome confirmed that both the core region and an adjacent duplicated domain share homologous MEIS1, PAX, and NF-Y motifs, reflecting a common origin in early osteichthyan history. Following this duplication, the two contiguous regulatory regions underwent distinct evolutionary trajectories: the ancestral core maintained strictly conserved basal motifs, whereas the duplicated segment experienced dynamic motif diversification across amniote lineages. Turtles and crocodilians acquired duplicated SMAD binding sites within the duplicated domain, whereas birds and mammals retained a single motif. Placental mammals and squamates accumulated additional NF-Y motifs, crocodilians and placental mammals gained supplementary CREB elements, and repeated PAX-binding sites independently expanded across multiple amniote orders, producing structurally complex, clade-specific regulatory modules capable of customized transcriptional responses.

Further interrogation of the amniote-specific PAX8 regulatory site revealed that it functions as an intricate composite regulatory node overlapping multiple adjacent transcription factor binding sites. Bioinformatic modeling of transcription factors predicted to bind this overlapping region revealed a statistically significant protein-protein interaction network characterized by an enrichment probability value of p = 8.28 × 10^-11. Functional enrichment profiling mapped divergent downstream physiological specializations between primary amniote lineages: the Sauropsida interaction network showed marked enrichment for endocrine system development, whereas the mammalian network displayed significant enrichment for thyroid hormone-mediated signaling cascades. Intriguingly, although the conserved genomic PAX8 recognition motif is maintained across avian genomes, the actual PAX8 protein-coding gene was lost during avian evolutionary history. The researchers concluded that selective pressure preserved the physical DNA motif in birds because alternative, related transcription factors belonging to the broader PAX protein family continue to recognize the nucleotide sequence and orchestrate hormone-responsive transcriptional regulation.

Phylogenetic rate calculations using the phyloP program under a neutral evolutionary model generated by phyloFit identified distinct episodic bursts of accelerated regulatory evolution across the vertebrate tree. Maximum likelihood phylogenetic reconstruction employing the K2P plus G4 substitution model revealed that promoter tree topology deviated from classical vertebrate species trees, underscored by extended branch lengths in vespertilionid bats and ancestral mammalian stems. Quantitative phyloP estimations confirmed statistically significant accelerated nucleotide substitution rates along the stem lineages of both Sauropsida and Mammalia, coinciding with rapid physiological diversification characterizing early amniote radiation. Furthermore, localized acceleration was detected along lineages leading directly to Aves, Rodentia, and Chiroptera. The researchers highlighted that two independent lineages exhibiting pronounced promoter acceleration, birds and bats, represent the only vertebrate clades that evolved powered flapping flight. Because flight demands substantial metabolic and biochemical remodeling of skeletal musculature, accelerated promoter evolution likely facilitated critical adaptive adjustments in Myostatin regulation and muscle growth control.

To directly test whether lineage-specific binding site differences alter embryonic gene expression, the researchers examined a variable microdomain located between the core TATA box and CREB motifs. In this intervening region, which contains an established LEF1/TCF site, avian promoters carry an ALX4 binding motif, whereas mammalian promoters possess a binding site for MYOD. Whole-mount and sectioned in situ hybridization in chicken and mouse embryos revealed a corresponding spatial compartmentalization of Myostatin transcripts during early development. In chicken embryos, Myostatin transcripts colocalized with ALX4 within the proliferative dermomyotome of the somites, whereas in mouse embryos, Myostatin expression coincided with MYOD within the differentiating myotome. This striking compartmental shift reflects differential recruitment by lineage-specific upstream regulators and may explain distinct developmental trajectories, such as robust muscle mass accumulation in avian species. The authors noted that future functional evaluations utilizing CRISPR-Cas9 base-editing or reciprocal promoter swaps in model organisms will provide experimental opportunities to interrogate these non-coding regulatory variations and assist genomic breeding strategies in livestock.

Subject of Research: Evolution and structural diversification of the vertebrate Myostatin promoter-enhancer cis-regulatory element

Article Title: Genetic mechanisms shaping the evolution of an ancient Myostatin promoter/enhancer across vertebrates

Article References: Mantovani, C. S., Metzker-Pinto, T., Buzzatto-Leite, I., Sampar, J. F., Fontoura, M. A., Jurberg, A. D., Nery, M. F., & Alvares, L. E. (2026). Genetic mechanisms shaping the evolution of an ancient Myostatin promoter/enhancer across vertebrates. Developmental Biology Advances, 17(1), Article 5. https://doi.org/10.1186/s13227-025-00256-4

Image Credits: AI Generated

DOI: 10.1186/s13227-025-00256-4

Keywords: Myostatin, cis-regulatory elements, comparative genomics, vertebrate evolution, promoter evolution, transcription factor binding sites, somite development, skeletal muscle, Genetic, mechanisms, shaping, evolution