DATA QUESTIONS. Carefully read the information below from a…
DATA QUESTIONS. Carefully read the information below from a recent research article. Then answer the following questions about the data presented. Distinct species of mammals, from mice to monkeys, have tails. However, humans and apes do not have tails (see Fig. 1). This loss of tail is thought to have contributed to the bipedalism in hominoids evolution. To understand the basis of tail loss during evolution, Xia and collaborators investigated the genomic variations in genes associated with tail development. Previous studies showed that mutations in the TBXT gene can cause short tails in mice. However, the genetic mechanism remained unknown. Fig. 1. Evolution of tail loss in hominoids. Tail phenotypes across the primatephylogenetic tree. Ma, millions of years ago. In this study, the researchers first characterized the sequence conservation and structure of the TBXT gene across primates. Below is the analysis of sequence conservation across distinct species of primates (Fig. 2). Shown is the structure of the human TBXT gene, with the exons depicted as color boxes. Alignment of homologous sequences in other primates is also shown (black boxes). They found that a specific repetitive element (AluY) was present in humans, as well as in great apes, but not in primates with tail. They also found another Alu element (AluSx1) that was present in all primates. Note that the AluY is in a reverse orientation of AluSx1. Fig. 2. Conservation of TBXT across primates. Exon sequences are shown in color boxes, the hominoid-specific AluY element is highlighted in red. The authors proposed that the insertion of the AluY element in hominoids could lead to the formation of a loop in the mRNA (formed by the pairing of AluY and AluSx1) that traps exon #6 (see Fig. 3). Then, this loop can alter the splicing of the TBXT mRNA leading to the loss of exon #6 in the mature mRNA, which could be linked to the loss of tail in hominoids. Fig. 3. Schematic of the proposed mechanism of tail-loss evolution inhominoids. To test their hypothesis, they used CRISPR to delete either AluY or AluSx1 in human stem cells. After confirming the deletions, they analyzed the resulting mRNA molecules by RT-PCR (Fig. 4). They analyzed normal cells (WT), cells with the AluY deleted (∆AluY), and cells with the AluSx1 deleted (∆AluSx1). Fig. 4. CRISPR deletions of TBXT sequence elements. a) CRISPR-generated homozygous deletions of the AluY element in TBXT intron 6 (top,) and AluSx1 element in intron 5 (bottom) in human stem cells. b) RT–PCR results of TBXT transcripts isolated from human ES cell of wild-type, TBXT∆AluY/∆AluY and TBXT∆AluSx1/∆AluSx1 genotypes. Finally, to test if exon #6 of TBXT is required for the tail development in other mammals they used CRISPR technologies to delete exon #6 in mouse embryos. After genome editing, the embryos were implanted into foster mothers and allowed to develop into pups (Fig. 5). Fig. 5. CRISPR deletions in mouse embryos. An heterozygous deletion of exon #6 of the TBXT gene in mouse embryos. Representative phenotypes of Tbxt+/+ and Tbxt∆exon6/+ mice are shown.
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