title: Geometric Morphometrics on Gene Expression Patterns Within Phenotypes: A Case Example on Limb Development.
abstract: How the genotype translates into the phenotype through development is critical to fully understand the evolution of phenotypes. We propose a novel approach to directly assess how changes in gene expression patterns are associated with changes in morphology using the limb as a case example. Our method combines molecular biology techniques, such as whole-mount in situ hybridization, with image and shape analysis, extending the use of Geometric Morphometrics to the analysis of nonanatomical shapes, such as gene expression domains. Elliptical Fourier and Procrustes-based semilandmark analyses were used to analyze the variation and covariation patterns of the limb bud shape with the expression patterns of two relevant genes for limb morphogenesis, Hoxa11 and Hoxa13. We devised a multiple thresholding method to semiautomatically segment gene domains at several expression levels in large samples of limb buds from C57Bl6 mouse embryos between 10 and 12 postfertilization days. Besides providing an accurate phenotyping tool to quantify the spatiotemporal dynamics of gene expression patterns within developing structures, our morphometric analyses revealed high, non-random, and gene-specific variation undergoing canalization during limb development. Our results demonstrate that Hoxa11 and Hoxa13, despite being paralogs with analogous functions in limb patterning, show clearly distinct dynamic patterns, both in shape and size, and are associated differently with the limb bud shape. The correspondence between our results and already well-established molecular processes underlying limb development confirms that this morphometric approach is a powerful tool to extract features of development regulating morphogenesis. Such multilevel analyses are promising in systems where not so much molecular information is available and will advance our understanding of the genotype-phenotype map. In systematics, this knowledge will increase our ability to infer how evolution modified a common developmental pattern to generate a wide diversity of morphologies, as in the vertebrate limb.
authors.affiliation: EMBL-CRG Systems Biology Program, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain; Universitat Pompeu Fabra (UPF), Barcelona, Spain;
authors.forename: Neus
authors.initials: N
authors.lastname: Martínez-Abadías
authors.affiliation: EMBL-CRG Systems Biology Program, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain; Universitat Pompeu Fabra (UPF), Barcelona, Spain;
authors.forename: Roger
authors.initials: R
authors.lastname: Mateu
authors.affiliation: EMBL-CRG Systems Biology Program, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain; Universitat Pompeu Fabra (UPF), Barcelona, Spain;
authors.forename: Martina
authors.initials: M
authors.lastname: Niksic
authors.affiliation: EMBL-CRG Systems Biology Program, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain; Universitat Pompeu Fabra (UPF), Barcelona, Spain;
authors.forename: Lucia
authors.initials: L
authors.lastname: Russo
authors.affiliation: EMBL-CRG Systems Biology Program, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain; Universitat Pompeu Fabra (UPF), Barcelona, Spain; Institució Catalana de Recerca i Estudis Avançats (ICREA), Pg. Lluís Companys 23, 08010 Barcelona, Spain james.sharpe@crg.eu.
authors.forename: James
authors.initials: J
authors.lastname: Sharpe
mesh_terms: D000818:Animals; D002965:Classification / Q000379:methods*; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000235:genetics; D018878:Limb Buds / Q000033:anatomy & histology / Q000196:embryology*; D051379:Mice; D010641:Phenotype
chemical_list: D018398:Homeodomain Proteins; C094041:Hoxa11 protein, mouse; C099959:homeobox protein HOXA13
keywords: Elliptical Fourier analysis; Hoxa genes; Procrustes-based semilandmark; gene domain; limb morphogenesis; whole-mount in situ hybridization