The absence of an external tail in humans and apes is a defining characteristic of hominoids, differentiating them from other primates. While the loss of the tail is hypothesized to have facilitated the evolution of bipedalism and orthograde posture, the underlying genetic mechanisms remained elusive until this study. Understanding the genetics of this trait offers insight into the evolutionary pressures shaping human traits. Previous research has identified numerous genes involved in tail development in other vertebrates, but pinpointing the specific genetic changes leading to tail loss in hominoids has proven challenging. The availability of primate genome sequences now allows for a more comprehensive investigation of genotype-phenotype relationships, paving the way for the identification of hominoid-specific genetic elements regulating tail development. This paper investigates the genetic basis for this evolutionary change, using comparative genomics and mouse models to explore the role of a specific genetic alteration in the *TBXT* gene.

Extensive research in developmental biology has illuminated the intricate gene regulatory networks governing tail development across vertebrates. The Mouse Genome Informatics (MGI) database catalogs over 100 genes associated with tail abnormalities, including genes such as *Tbxt*, *Wnt3a*, and *Msgn1*. These genes play crucial roles in mesoderm and definitive endoderm formation, essential processes during embryonic development. Perturbations in these genes can result in shortened or absent tails. However, the specific genetic alterations driving tail loss in hominoids remained unknown until the present study, which focused on identifying hominoid-specific variants potentially linked to the phenotype. The study utilized information from existing databases and examined both coding and non-coding regions of genes related to tail development, searching for hominoid-specific variants that could explain the observed phenotype. The study further draws upon previous work on primate genome sequencing, which is helping to uncover the genetic basis of numerous phenotypic traits.

The researchers employed a multi-faceted approach to investigate the genetic basis of tail loss. They began by screening 31 human genes, along with their primate orthologues, known to be associated with tail development in other species. This initial screen focused on protein-coding regions. Upon failing to identify strong candidates, they expanded their search to include 109 additional genes linked to tail reduction in mice and systematically screened the entire genomic regions of these genes, extending 10 kb upstream and downstream. This broad search resulted in the identification of numerous single nucleotide variants (SNVs), deletions, and insertions specific to hominoids. Further analysis focused on non-coding hominoid-specific variants, leading to the identification of an Alu element insertion within the sixth intron of the *TBXT* gene. This Alu element, belonging to the AluY subfamily, exhibited a hominoid-specific phylogenetic distribution and is located near another Alu element in the reverse orientation. The researchers hypothesized that the AluY insertion facilitated alternative splicing of *TBXT*, creating a shorter isoform missing exon 6. To test this hypothesis, they generated several mouse models: a heterozygous model expressing both full-length and exon-skipped *Tbxt* isoforms and a homozygous model that aimed to recapitulate the alternative splicing event observed in humans using different approaches. These included using CRISPR-Cas9 gene editing in human and mouse embryonic stem cells to remove the Alu elements, generating different transcripts and allowing the investigation of the alternative splicing patterns and their effects on tail development. The resulting mouse models were characterized for their tail morphology, and the expression patterns of *Tbxt* isoforms were analyzed using RT-PCR. Transcriptomic analyses were also performed to assess the effects of the *TBXT* isoforms on downstream gene expression. These results were further substantiated by using a variety of techniques, including Sanger sequencing to confirm the desired modifications, and RNA structure prediction to support the alternative splicing mechanism. The various analyses were carried out using many bioinformatic tools as well as experimental models.

The study found a hominoid-specific AluY element insertion in the sixth intron of *TBXT*. This insertion, in conjunction with a pre-existing AluSx1 element, leads to alternative splicing resulting in a *TBXT* isoform lacking exon 6 (*TBXT*Δexon6). Deletion of either Alu element in human embryonic stem cells abolished the production of the shorter isoform, confirming their interaction in promoting alternative splicing. In mouse models, expression of the exon-skipped *Tbxt* isoform (*Tbxt*Δexon6) was sufficient to induce tail-loss or shortening phenotypes, with the severity correlating with the relative abundance of the shorter isoform compared to the full-length isoform. The heterozygous mouse model (*Tbxt*Δexon6/+) showed incomplete penetrance of the phenotype, while homozygous *Tbxt*Δexon6/Δexon6 embryos were non-viable and exhibited neural tube defects. Further experiments with mouse models where AluY and AluSx1 elements, or their equivalent reverse complementary sequences (RCS), were inserted into the mouse *Tbxt* gene demonstrated that the presence of inverted repeated sequences was sufficient to induce alternative splicing and a shortening of the tail. The relative expression levels of the *Tbxt* full-length and the *Tbxt*Δexon6 isoforms played a crucial role in determining tail length. Homozygous deletion of exon 6 in mice proved lethal, indicating a crucial role for the full-length *Tbxt* isoform in development. These findings strongly suggest that the AluY insertion in *TBXT* is a significant contributor to tail loss in hominoids. The RNA sequencing data revealed differential expression of *Tbxt* target genes across the various mouse models.

The findings provide compelling evidence for a novel mechanism driving tail-loss evolution in hominoids. The AluY insertion in *TBXT*, rather than directly affecting splicing signals, interacts with a neighboring Alu element to alter splicing patterns. This highlights the ability of transposable elements to significantly impact gene function and contribute to phenotypic evolution. The mouse models convincingly demonstrate the sufficiency of the *Tbxt*Δexon6 isoform in inducing tail loss or reduction. The incomplete penetrance in the heterozygous model likely reflects the complex interplay of genetic and environmental factors during tail development. The observed lethality of homozygous exon 6 deletion and the occurrence of neural tube defects highlight a potential evolutionary trade-off: the selective advantage of tail loss may have come at the cost of increased susceptibility to neural tube defects. This trade-off may continue to impact human health today, as mutations in *TBXT* have been linked to neural tube defects and sacral agenesis. The identification of the Alu element insertion in the *TBXT* gene provides valuable insight into the genetic events shaping hominoid evolution.

This study elucidates a crucial role for a hominoid-specific AluY insertion in *TBXT* in causing tail-loss through alternative splicing. Mouse models confirm that the resulting shorter *Tbxt* isoform is sufficient to cause tail loss but also introduces a risk of neural tube defects, signifying a potential evolutionary trade-off. Future research could focus on investigating the detailed molecular mechanisms by which the *TBXT*Δexon6 isoform affects gene regulation and exploring the potential contributions of additional genetic changes in stabilizing the no-tail phenotype in hominoids. The identification of similar mechanisms in other primate lineages with independent tail loss would further strengthen the understanding of convergent evolution.

The study mainly focuses on the role of the *TBXT* gene and the identified Alu insertion. It acknowledges that additional genetic factors may have contributed to the complete loss of the tail. The incomplete penetrance of the tail phenotype in the heterozygous mouse model may limit the straightforward interpretation of the results. The generation of the *Tbxt*insASAY mouse model did not produce high levels of the *Tbxt*Δexon6 isoform in the embryonic tailbud, possibly because of differential splicing regulations in different cell types. Further investigations are needed to fully understand the complex regulatory network involved in tail development and the precise role of the *TBXT* isoforms.