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'Sonic Hedgehog' alters body shape
The difference between webbed toes and the distinct shape of animal toes that we know, may not be the result of just genetic information — but how genes regulate information. Researchers at the National Institute of Genetics, Research Organization of Information and Systems (ROIS) in Japan found that a small, non-specific tweak to a mammal's DNA can potentially cause significant and very specific, physical changes. Their work was recently published in the Proceedings of the National Academy of Sciences or the PNAS.
"We aim to illustrate that a change in regulatory information of a certain gene definitely triggers change of limb shape."
Shiroishi and his team examined a mutant mouse line, dubbed "Hammer toe," that appeared about 50 years ago. Instead of well-defined digits, these mice have webbed and sometimes fused toes. Previous studies revealed the mutation resulted from the mis-regulation of a protein named Sonic hedgehog (shh). Shh is responsible for cell differentiation. According to Shiroishi, past analyses explain polydactylism — extra digits — but not the webbing and fusion found in Hammer toe mice.
"Initially, we thought they might be the same type of mutation, and looked at the enhancer," Shiroishi explains, referring to a piece of DNA that promotes expression of proteins that enhance specific physical characteristics. "But actually, the Hammer toe mutation is a large insertion between the enhancer and Sonic hedgehog."
Researchers found a non-coding DNA fragment from chromosome 14 had become inserted into chromosome five located near the Sonic hedgehog gene. This helps explain the unique Hammer toe syndactylyism (Greek meaning "together" and "finger") as opposed to a mutation like polydactylism (Greek (polys), meaning 'many' and (daktylos), meaning 'finger').
Shiroishi: "In the cell nucleus, a number of transcription factors interact with the genome to orchestrate gene expression. The addition of new information means that the gene receives a new access point for transcription factors. If the transcription factor is an activator, it would raise expression levels of that gene."
The spontaneous mutation found in Hammer toe may also echo convergent evolution seen across many species. Similar morphology is seen in the webbed feet of frogs, water birds, even the South American capybara, the world's largest water loving rodent. Examples of independent species evolving similar traits by adapting to similar environments, Shiroishi points out, may reflect the same shh enhancer as seen in the Hammer toe mouse, becomming activated and developing into, perhaps, bat wings.
Shiroishi: ""Diversity of organ shape among vertebrate animals is controlled by differential gene regulation ... interchromosomal translocation generates enhancer activity for a developmental gene, ultimately leading to morphology alteration. During evolution, bats have acquired regulatory information for genes specifically activated in their wings. We would like to identify convergence-related enhancers in animals, and clarify how regulatory evolution impacts their morphology."
In this study, we reexamined an old mouse mutant named Hammer toe (Hm), which arose spontaneously almost a half century ago and exhibits a limb phenotype with webbing. We revealed that a 150-kb noncoding genomic fragment that was originally located in chromosome 14 has been inserted into a genomic region proximal to Sonic hedgehog (Shh), located in chromosome 5. This inserted fragment possesses enhancer activity to induce Shh expression in the interdigital regions in Hm, which in turn down-regulates bone morphogenetic protein signaling and eventually results in syndactyly and web formation. Since the donor fragment residing in chromosome 14 has enhancer activity to induce interdigital gene expression, the Hm mutation appears to be an archetypal case of enhancer adoption.
Acquisition of new cis-regulatory elements (CREs) can cause alteration of developmental gene regulation and may introduce morphological novelty in evolution. Although structural variation in the genome generated by chromosomal rearrangement is one possible source of new CREs, only a few examples are known, except for cases of retrotransposition. In this study, we show the acquisition of novel regulatory sequences as a result of large genomic insertion in the spontaneous mouse mutation Hammer toe (Hm). Hm mice exhibit syndactyly with webbing, due to suppression of interdigital cell death in limb development. We reveal that, in the Hm genome, a 150-kb noncoding DNA fragment from chromosome 14 is inserted into the region upstream of the Sonic hedgehog (Shh) promoter in chromosome 5. Phenotyping of mouse embryos with a series of CRISPR/Cas9-aided partial deletion of the 150-kb insert clearly indicated that two different regions are necessary for the syndactyly phenotype of Hm. We found that each of the two regions contains at least one enhancer for interdigital regulation. These results show that a set of enhancers brought by the large genomic insertion elicits the interdigital Shh expression and the Hm phenotype. Transcriptome analysis indicates that ectopic expression of Shh up-regulates Chordin (Chrd) that antagonizes bone morphogenetic protein signaling in the interdigital region. Indeed, Chrd-overexpressing transgenic mice recapitulated syndactyly with webbing. Thus, the Hm mutation provides an insight into enhancer acquisition as a source of creation of novel gene regulation.
Authors: Kousuke Mouri, Tomoko Sagai, Akiteru Maeno, Takanori Amano, Atsushi Toyoda and Toshihiko Shiroishi
The authors declare no conflict of interest.
We thank Y. Mizushina, K. Fukunaga, A. Kondo, Y. Suzuki, and H. Nakazawa for generating transgenic mice and mutants; Dr. A. McMahon for the Shh riboprobe and Dr. R. Hill for the Msx2 riboprobe; and the staff of Comparative Genomics Laboratory at NIG for supporting genome sequencing. This study was supported by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS) (Grants JP15J06985, JP17K15162, and JP17K19411).
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X-ray micro-CT scans show that the Sonic hedgehog gene is functioning
in the the area between digits, causing interdigital webbing or 'Hammer toe.'
Image credit: National Academy of Sciences of the United States of America.