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3D GENE SHAPE Report Summary

Project ID: 327382
Funded under: FP7-PEOPLE
Country: Spain

Final Report Summary - 3D GENE SHAPE (Hearts and limbs: Linking morphometrics with 3D analysis of gene expression patterns)

Despite great investment in developmental biology research to understand how organs are formed and how disease can affect normal developmental process, we still do not have a highly accurate methodology that allows us to pinpoint how, when and which processes of development are altered during embryonic development to produce birth malformations. To overcome this issue, we proposed to combine innovative 3D imaging and shape analysis tools: Optical projection tomography (OPT) microscopy and Geometric Morphometrics (GM).

OPT is an imaging technique ideally suited for 3D imaging of developing embryonic structures from 1 to 10 mm across. The great advantage of OPT imaging is that it can be used with experimental samples previously processed using genetic engineering and/or molecular techniques to visualize where and how much a certain gene is expressed in a certain moment of development (Fig. 1). This allows for the mapping of genes within a tissue in a true three-dimensional framework. By switching on and off, genes provide the detailed instructions to build an organism. Therefore, it is absolutely crucial to know when, where and how genes are expressed at every moment of development. OPT offers the technology to perform such research, providing invaluable ways to visualize gene expression, at least in model organisms such as mice.

However, methods for quantifying these 3D expression distributions in a systematic, objective manner are lacking. For this reason, we proposed to apply Geometric Morphometrics (GM), a sophisticated body of robust statistical tools that have been developed for measuring and comparing 3D shapes with increased precision and efficiency. GM has found wide application in the biological sciences to analyze anatomical data, causing an authentic “revolution” in the morphometric field in the last decades, but has not yet been applied to the shape analysis of gene expression domains.

The main objective of our project is thus to develop a novel 3D quantitative approach to the analysis of gene expression domains in association with developing organs combining OPT and GM (Fig. 2). To demonstrate its relevance in biomedical research, we have used a mouse model of a congenital disease called Apert syndrome. Apert syndrome is a rare disorder characterized by malformations in the skull, brain, heart, limbs and lungs. Over 99% of Apert cases are caused by one of two mutations in the FGFR2 (Fibroblast Growth Factor Receptor 2) gene. Patients carrying the P253R mutation show more severe limb malformations, such as fusion of digits. Our specific aims are:

Aim 1.- Apply the OPT-GM approach for characterizing the size and shape of the heart and the limb in Apert syndrome. Using control and mutant littermates of the Apert syndrome Fgfr2+/P253R mouse model at two different time points of development (embryonic days E10.5 and E11.5), we will visualize and quantify the size and shape of embryonic structures affected by Apert syndrome, such as the limb and the heart, jointly with the expression patterns of two genes, Dusp6 and Hand2, known to be affected by the FGFR2 mutation causing Apert syndrome (Fig. 3).

Aim 2.- Establish spatio-temporal interactions between structures and gene expression patterns in normal and altered development in Apert syndrome. We will exemplify how using our OPT-GM approach it can be assessed whether a disease can alter the spatio-temporal map of interactions between the shape of an organ and the shape of gene expression domains relevant for its development by following the dynamic spatio-temporal changes in gene expression patterns in the limb and the heart of control and mutant littermates of the Apert syndrome Fgfr2+/P253R mouse model.

Aim 3.- Identify altered developmental processes in Apert syndrome. Statistical analyses comparing the size and shape of the hearts and the limb of control and mutant littermates mouse will reveal the stage at which normal development is altered in Apert syndrome.

• Main results achieved
OPT imaging successfully revealed the expression patterns of Dusp6 and Hand2 within whole embryos at 10.5 and 11.5 embryonic days in a 3D framework. Size analyses showed that mutant Fgfr2+/P253R (MUT) mice have larger limbs at early stages but smaller limbs at later stages in comparison to control (WT) mice. Regarding the volumes of the gene expression patterns, the domains were larger in MUT than in WT mice, but the differences were only significant for Hand2. Geometric morphometric analyses of 3D landmark-based data showed that the spatiotemporal changes during limb development were highly dynamic and different between forelimbs and hindlimbs, and between WT and MUT littermates. Morphological differentiation started as early as embryonic day 10 and was coupled with a different expression pattern of both Dusp6 and Hand2 expression domains. Despite the Fgfr2 P253R mutation induces changes in the shape of the limbs and their associated gene expression patterns, the covariation pattern between the limb and the gene expression domains were not disrupted. Regarding the heart, we found that the most affected region was the right ventricle, but no significant differences in the cardiac chambers’ size were found between WT and MUT littermates, except for an increased Dusp6 expression in the left ventricle of MUT littermates between E10.5 and E11. In conclusion, our results demonstrate that our method combining OPT and GM is an accurate and insightful tool to compare normal and disease-altered patterns of variation and to reveal how the genotype translates into the phenotype. Precise embryonic phenotyping of Apert syndrome mice helped us to pinpoint that altered Fgf/Fgfr signalling has direct consequences on target genes that contribute to limb and heart malformations as early as E10.5.

• Expected final results and their potential impact and use
Our method combining OPT and GM has proved an accurate and insightful tool to compare normal and disease-altered patterns of variation and to reveal how the genotype translates into the phenotype. Precise embryonic phenotyping of Apert syndrome mice helped us to pinpoint when and which developmental processes were altered by the disease. These results will help us further understand the origins of abnormal limb and heart morphogenesis in Apert syndrome. If our method could be further developed and automatized, it would turn into high-through output promising tool for biomedical research, providing insight into the processes that cause malformations and lead to malfunction and disease. This is essential for understanding diseases and discovering potential therapies.

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