Identifying and Characterising Gene Regulatory Changes in Human Skeletal Evolution and Disease Using Vertebrate Models

Lead Research Organisation: MRC London Institute of Medical Sciences

Abstract

Skeletal development is a fundamental biological process with a major impact on human health. Defects in it cause many human congenital syndromes that affect bone density, bone turnover or bone structure, leading to debilitating bone fractures. Yet, the genetic mechanisms that instruct each and every one of the 206 human bones to develop into a unique shape at a unique anatomic location are largely unknown. To uncover these mechanisms, we are taking advantage of the vast differences in bone shape seen in nature to identify molecular events that lead to skeletal change. We use genetic crosses between threespine stickleback populations with dramatic skeletal differences to identify key genes regulating skeletal development. We are also comparing the human and other primate genomes to identify regions that surprisingly differ in their sequence. In most cases, we find that these fall in non-coding regions, which contain gene regulatory elements that switch genes on and off in spatial and temporal manner. Using mouse model to recreate the sequence changes in the regulatory elements, we are asking whether these sequence differences can account for human-specific skeletal modifications. Gene regulatory elements that have been altered during vertebrate evolution to modify bone growth are likely to be indispensible for bone development, and our findings should provide candidate genomic regions to screen for mutations in patients with unknown cause of skeletal disease.

Technical Summary

Humans differ from other primates in a wide range of traits, including dramatic modifications of the bones in the skeleton. Skeletal development is a fundamental biological process that has a major impact on human health, yet the genetic mechanisms that instruct each and every one of the 206 human bones to develop into a unique shape at a unique anatomic location are largely unknown. To complement our current knowledge, we use a multipronged approach that combines work in two experimental systems (threespine stickleback and mouse), and comparative genomics of humans and other species to identify molecular events that correlate with skeletal evolution.

Threespine sticklebacks are particularly well suited to genetic studies of evolution, because they have undergone one of the most recent and best-documented examples of vertebrate adaptive radiation. Marine sticklebacks typically have robust skeletons and body armour that have been dramatically reduced in many freshwater populations throughout the Northern hemisphere. We have previously used a full suite of genetic, genomic, and transgenic tools to map armour plate size to a key skeletal development gene, called Gdf6. Mutations in the coding regions of the human homolog of this gene cause Klippel-Feil syndrome, with patients exhibiting skeletal and other abnormalities. Freshwater fish do not have coding changes in the Gdf6 gene, and instead allele-specific expression studies revealed gene regulatory changes. Armour plate size is one of numerous other skeletal traits that have evolved in parallel in many freshwater stickleback populations. For the ones that have been mapped so far we see a striking evolutionary pattern. Gene regulatory changes in key developmental control genes are a predominant cause of repeated evolution.

In parallel to the stickleback studies, we have previously surveyed the human genome for the types of regulatory mutations that control much of the stickleback evolution. We identified a set of over 500 deletions in the human genome that remove sequences otherwise highly conserved between chimpanzee and other mammals. Almost all of them occurred in gene regulatory regions. We are cloning the ancestral region from a closely related outgroup where the sequence is still present (chimpanzee) and we are asking whether are they driving expression in the developing mouse skeleton at anatomic sites that have changed during human evolution.

To complement the unbiased genome-wide approaches, we have also initiated a targeted survey with the long-term goal of identifying the entire gene regulatory landscape surrounding Runx2, a key skeletal development gene. This gene is already known to have dosage sensitive effects on midface size and prognathism in cleidocranial dysplasia patients. However, about a third of the patients with this condition do not have coding changes in Runx2. Our survey has already identified regions that drive expression in sites with striking anatomical changes in the human lineage. We plan to test the role of particular enhancer changes, by using the mouse to recreate sequence changes that are specific to the human lineage. Future work will determine whether these regulatory regions are also mutated in human disease.

Publications

10 25 50
 
Title RUNX2 LacZ and knockout/knockin lilnes 
Description These mouse lines are allowing us to study the function of enhancers in the RUNX2 locus that shape bone development. 
Type Of Material Model of mechanisms or symptoms - human 
Provided To Others? No  
Impact A manuscript about the use of these lines is in preparation and once published will highlight enhancers in the RUNX2 locus that may have mutations in human patients with Cleidocranial dysplasia. 
 
Title Sticklebacks as model for skeletal development and evolution 
Description Sticklebacks are a great system for identifying the genetic basis of skeletal traits and performing the discovery stage in the stickleback (fish) system reduces the number of mouse we have to use. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact We have identified changes in a stickleback GDF6 enhancer to be responsible for the changes in bone development in that species, and subsequently we looked to see whether the regulatory region of this gene has changed as well in humans. We identified an ancestral conserved enhancer in the GDF6 locus that has been deleted in humans that likely played a role in our transition from arboreal to bipedal ape. This example illustrates how we used the stickleback model to help us inform a screen done to identify genetic basis of human-specific traits. 
URL http://www.cell.com/cell/abstract/S0092-8674(15)01631-1
 
Description RUNX2 enhancer modifications in the human lineage 
Organisation Stanford University
Department Department of Developmental Biology
Country United States 
Sector Academic/University 
PI Contribution I am analysing the expression patterns of LacZ mouse lines and making mouse knockout and knockin lines to test the function of enhancers.
Collaborator Contribution They helped with the making of the LacZ mouse lines and with computational analysis that led to the identification of an interesting facial enhancer.
Impact We are currently assembling the collected data and a manuscript is in preparation.
Start Year 2010
 
Description Cambridge University visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact I presented my research on identifying the genetic basis of skeletal traits, which led to interesting questions and discussions.
Year(s) Of Engagement Activity 2015
 
Description Evolution 2016 Conference, Austin, TX, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact About 100 scientists attended my talk, which sparked questions and discussion afterwards.
Year(s) Of Engagement Activity 2016
 
Description Evolution of Gene Regulation Conference, London, UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I presented my research on gene regulation, which led to many questions and stimulating discussion afterwards.
Year(s) Of Engagement Activity 2014
 
Description Evolutionary Genetics and Genomics Symposium 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact About 100 scientists attended the conference, which sparked questions and discussion aferwards.
Year(s) Of Engagement Activity 2006,2016
 
Description Oxford University Visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact I presented my research using stickleback and human models for skeletal evolution, which led to many questions and interesting discussions afterwards.
Year(s) Of Engagement Activity 2014
 
Description The 49th Population Genetics Group Meeting, Edinburgh, UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I presented my research on the role of bone morphogenetic proteins in skeletal evolution, which led to many questions and stimulating discussions afterwards.
Year(s) Of Engagement Activity 2015
 
Description The European Human Genetics Conference 2016, Barcelona, Spain 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact About 100 scientists attended my talk, which sparked questions and discussion afterwards.
Year(s) Of Engagement Activity 2016
 
Description The European Molecular Biology Laboratory Visit, Heidelberg, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I presented my work on the genetics of skeletal traits, which led to thought-provoking discussions afterwards.
Year(s) Of Engagement Activity 2015
 
Description The Society for Molecular Biology and Evolution Conference, Vienna, Austria 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I presented my work on gains and losses of enhancers in vertebrate skeletal evolution, which led to stimulating discussion afterwards.
Year(s) Of Engagement Activity 2015
 
Description University College London Visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact I presented my work, which led to stimulating discussions about my area of research.
Year(s) Of Engagement Activity 2014
 
Description University of Leeds Visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact I presented my research on stickleback and human evolution, which led to stimulating discussions afterwards.
Year(s) Of Engagement Activity 2015
 
Description University of Leicester Visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other audiences
Results and Impact I presented my research on skeletal traits evolution, which led to a great questions and discussion afterwards.
Year(s) Of Engagement Activity 2014