The question of how vertebrates—the group that includes fish, amphibians, reptiles, birds, and mammals—evolved their remarkable complexity compared to their invertebrate ancestors has long fascinated evolutionary biologists. Now, an international team of researchers has identified a striking pattern in how certain critical genes behave differently in vertebrates versus invertebrates, potentially solving a piece of this evolutionary puzzle.

The study, published in BMC Biology, used cutting-edge long-read DNA sequencing technology to examine gene expression during embryonic development in three species representing key evolutionary transitions: the sea squirt Ciona intestinalis (an invertebrate closely related to vertebrates), the European brook lamprey Lampetra planeri (a jawless vertebrate), and the Western clawed frog Xenopus tropicalis (a jawed vertebrate).

“It was very surprising to us to see how this small selection of very particular genes stands out in the way that they are behaving compared to any other sort of gene we looked at,” said lead author Professor David Ferrier from the University of St Andrews. “It will be exciting to determine how these various different protein forms work in distinct ways to generate the diversity of cell types we now see in vertebrates.”

The genes in question encode transcription factors—proteins that control which other genes get turned on or off—that serve as the final output of three major cell signaling pathways: the Wnt/β-catenin, Hedgehog, and BMP pathways. These ancient communication systems are fundamental to animal development, orchestrating everything from establishing body axes to forming organs.

The Isoform Advantage

At the heart of the discovery lies the concept of transcript isoforms. A single gene can produce multiple slightly different versions of its protein product through a process called alternative splicing. These variations, or isoforms, can have distinct functions, allowing organisms to extract more functional diversity from the same number of genes.

The researchers found that in vertebrates, the genes encoding TCF/LEF proteins (the main transcription factors of Wnt signaling), SMAD proteins (mediators of BMP signaling), and GLI proteins (effectors of Hedgehog signaling) produce significantly more isoforms during development than in the invertebrate sea squirt. Importantly, this pattern was not seen in other categories of genes, including other transcription factors, other components of these same signaling pathways, or even the closely related SOX gene family.

In Ciona, the researchers found just one TCF/LEF gene producing one transcript, compared to one gene producing two transcripts in lamprey and four genes producing nine transcripts in the frog. Similar expansions were observed for SMAD and GLI genes.

This finding addresses what scientists call the “G-value paradox”—the observation that organism complexity does not correlate with the number of genes in a genome. Humans, for example, have roughly the same number of genes as the nematode worm C. elegans. Previous research had suggested that alternative splicing might help resolve this paradox by expanding the functional repertoire of proteins without requiring more genes. The new study demonstrates that this expansion appears to have been concentrated in specific, functionally critical genes.

The Wnt, Hedgehog, and BMP signaling pathways are not just important for normal development—they are also frequent targets of disease-causing mutations and major focuses of pharmaceutical research. Disruptions in Wnt signaling have been linked to various cancers and diabetes, while Hedgehog pathway abnormalities contribute to developmental disorders and certain tumors.

Previous research had established a strong correlation between alternative splicing and the number of cell types in an organism, which serves as a proxy for complexity. However, whether particular types of genes contributed disproportionately to this phenomenon had not been assessed until now.

The researchers used linear regression modeling to compare how transcript-to-gene ratios changed across species for different gene categories. When they grouped TCF/LEF, SMAD, and GLI genes together and compared them to all other gene categories, the signaling transcription factors showed a significantly steeper increase in isoform diversity from invertebrates to vertebrates.

This pattern runs counter to a general evolutionary principle. Previous studies had found that gene duplication and isoform diversity tend to be inversely correlated—duplicated genes usually retain fewer isoforms. The signaling transcription factors studied here appear to be exceptions, having both duplicated at the origin of vertebrates (likely through whole-genome duplications) and maintained or expanded their isoform diversity.

New Discovery in an Old Model Organism

The study also yielded an unexpected bonus finding. While confirming the structure of the TCF/LEF gene in Ciona intestinalis, the researchers discovered a previously unknown splice isoform specific to this species, produced only after the gastrulation stage of embryonic development.

Analysis revealed that this new exon arose through the insertion of transposable elements—mobile genetic sequences that can jump around genomes—combined with point mutations that created new splice acceptor sites and stop codons. This provides a concrete example of how transposable elements can contribute to evolutionary innovation by creating new protein variants.

The team’s approach of using long-read sequencing technology was crucial to these discoveries. Traditional short-read sequencing struggles to accurately determine which exons belong together in a single transcript, making isoform identification unreliable. Long-read methods capture entire transcripts in single reads, providing definitive isoform structures.

Implications for Understanding Vertebrate Evolution

The findings suggest that increased diversity in these master regulatory proteins may have played a disproportionate role in enabling the evolution of vertebrate complexity. While vertebrates underwent whole-genome duplications early in their evolution, these events alone cannot explain why vertebrates became so much more complex than their invertebrate relatives. The targeted expansion of functional diversity in key developmental regulators offers a compelling piece of the puzzle.

The researchers acknowledge limitations—their analysis focused on developmental stages and may not capture all isoforms expressed throughout an organism’s life. Additionally, while Ciona is the closest invertebrate relative to vertebrates, some aspects of its genome are derived, meaning some findings may require validation in other invertebrate species.

Nevertheless, these long-read transcriptomes represent valuable new resources for understanding gene expression during chordate development, particularly for species like lampreys and sea squirts that had not previously been studied with this technology.

Future research will need to determine exactly how these different protein isoforms function differently to generate the diversity of cell types seen in vertebrates—work that could have implications not only for understanding evolution but also for developing therapies targeting these fundamental signaling pathways.

Endnotes

1. Torres-Aguila NP, Salonna M, Shimeld SM, Hoppler S, Ferrier DEK. Long-read sequencing reveals increased isoform diversity in key transcription factor effectors of intercellular signalling at the invertebrate-vertebrate transition. BMC Biology. 2026;24:28. doi:10.1186/s12915-026-02522-w
2. University of St Andrews. New discovery sheds light on evolutionary crossroads of vertebrates. EurekAlert! February 1, 2026. https://www.eurekalert.org/news-releases/1114438
3. Chen L, Bush SJ, Tovar-Corona JM, Castillo-Morales A, Urrutia AO. Correcting for differential transcript coverage reveals a strong relationship between alternative splicing and organism complexity. Molecular Biology and Evolution. 2014;31:1402-13.
4. Hahn MW, Wray GA. The g-value paradox. Evolution & Development. 2002;4:73-5.
5. Bush SJ, Chen L, Tovar-Corona JM, Urrutia AO. Alternative splicing and the evolution of phenotypic novelty. Philosophical Transactions of the Royal Society B. 2017;372:20150474.
6. Hoppler S, Waterman ML. Evolutionary Diversification of Vertebrate TCF/LEF Structure, Function, and Regulation. In: Wnt Signaling in Development and Disease. Hoboken, NJ: John Wiley & Sons; 2014. p. 225-37.
7. Kopelman NM, Lancet D, Yanai I. Alternative splicing and gene duplication are inversely correlated evolutionary mechanisms. Nature Genetics. 2005;37:588-9.

IMAGE SOURCE: Steve Lonhart / NOAA MBNMS