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Session Schedule & Abstracts

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Friday 1st July, 2016

Symposium: Fundamental aspects of the spatial associations, development, and birth defects of the muscles and skeleton in non-pentadactyl limbs 1

Room: Salon H   9:30 am–11:00 am

Moderator(s): V. Abdala, T. Kohlsdorf, & R. Diogo
NPL1-1  9:30 am  Non-pentadactyly, soft and hard tissue associations, birth defects, and implications for medicine. Smith C.*, Howard University; Diogo R., Howard University
Abstract: Signaling for limb bone development usually precedes that for muscle development, such that cartilage is generally present before muscle formation. It remains obscure, however, if: (i) tetrapods share a general, predictable spatial correlation between bones and muscles; and, if that is the case, if (ii) such a correlation would reflect an obligatory association between the signaling involved in skeletal and muscle morphogenesis. We address these issues here by using the results of a multidisciplinary analysis of the appendicular muscles of all major tetrapod groups integrating dissections, muscle antibody stainings, regenerative and ontogenetic analyses of fluorescently-labeled (GFP) animals, studies of non-pentadactyl human limbs related to birth defects, mathematical insights concerning for instance the possible display of a Turing-model type of mechanism associated with digit formation, and new, state-of the art systems biology tools such as anatomical network analyses. Our synthesis suggests that there is a consistent, surprising anatomical pattern in both normal and abnormal phenotypes, in which the identity and attachments of distal limb muscles are mainly related to the topological position, and not to the developmental primordium (anlage) or even the homeotic identity, of the digits to which they are attached, thus providing a starting point towards the resolution of a centuries-old question raised by authors such as Owen about the specific associations between limb bones and muscles. This question has crucial implications for evolutionary and developmental biology, and for human medicine because non-pentadactyly is the most common birth defect in human limbs. In particular, this synthesis paves the way for future developmental experimental and mechanistic studies, which are needed to clarify the processes that may be involved in the elaboration of the anatomical patterns described here, and to specifically test the hypothesis that distal limb muscle identity/attachment is mainly related to digit topology.

NPL1-2  10:00 am  The genetic basis of mammalian limb diversification. Sears KE*, University of Illinois; Maier JA; Rivas-Astroza M; Cao X; Zhong S; Zhao K; Sinha S; Ma J; Behringer R; Cretekos C; Rasweiler J
Abstract: From bat wings to horse hooves, mammalian limb diversification has been crucial to the evolutionary success of the group. Indirect evidence from studies of mammalian limb evolution suggests that mammalian limb diversification, including the frequent limb reduction that characterizes many mammalian groups, has not occurred primarily by the evolution of new genes, but by differential regulation of existing genes shared by all mammals and inherited from an ancestral genetic toolkit. However, the specific genes and regulatory mutations that are responsible for limb diversification remain unknown for most mammalian groups. To begin to identify these genes and regulatory mutations, we used RNASeq to compare the transcriptomes of the developing limbs of several mammals, including pentadactyl mice, bats, and opossums, and tetradactyl pigs. Results suggest that gene expression varies more during later than earlier stages of limb development, both within and among species. Consistent with this, results suggest that the evolutionary age of each species' transcriptome decreases as developmental age increases. Within the more variable, later-expressed genes, we identified significant differences in the expression levels of HoxA and HoxD genes (N = 9) within and among species. WISH generally confirmed these RNASeq results, and uncovered key differences in expression domains as well. We used computational approaches to identify candidate enhancers for HoxA and HoxD genes, and functionally tested candidate enhancers using in vitro luciferase assays. Through this approach we identified several candidate enhancers with the potential to drive lineage-specific Hox expression levels. Taken together, our results support the hypothesis that the hierarchical nature of development translates into increasing variation as development progresses, and that divergence of Hox gene expression during these later stages of development plays a role in mammalian limb diversification.

NPL1-3  10:30 am  What determines the identity of the distal limb muscles? A myological analysis of mammals with digit reduction/digit loss. Bello-Hellegouarch G*, Department of Biology, FFCLRP, University of São Paulo, Avenida Bandeirantes, 3900, Bairro Monte Alegre, Ribeirão Preto, São Paulo, Brazil; Diogo R, Department of Anatomy, Howard University College of Medicine, Washington, District of Columbia, 20059; Abdala V, Instituto de Biodiversidad Neotropical, CONICET-UNT, Miguel Lillo 251, Tucumán, Argentina; Kohlsdorf T, Department of Biology, FFCLRP, University of São Paulo, Avenida Bandeirantes, 3900, Bairro Monte Alegre, Ribeirão Preto, São Paulo, Brazil
Abstract: Using the pentadactyl limb as a reference, muscles and bones are usually superimposed in a sharp spatial association. But does this spatial correlation reflect an obligatory association during morphogenesis? The somatic limb muscle progenitor cells apparently do not carry intrinsic positional information; the presence of the first tissue formed during limb development (condensations that will give rise to bones) may provide the positional signaling for the subsequent development of the muscles. According to this scenario, it can be expected that in non-pentadactyl limbs bones and muscles are still spatially superimposed. How the loss of bony structures affects muscle anatomy? Does the muscle reduction parallel the bone reduction in non-pentadactyl limbs? Addressing these questions will help us clarify the specific correlation existing between limb bones and muscles. However, the studies of limb development and evolution have emphasized skeletal tissues, while soft tissues such as muscles have been neglected. With those questions in mind, we dissected representatives of the main groups of mammals with different grades of digit reduction or digit loss to test the hypothesis that the identity and configuration of the autopodial muscles is mainly related to the topological position of the digits to which the muscles attach. Our results suggest that there is a consistent, anatomical pattern in which the identity of the muscles of the autopodium are mainly related to the topological position, and not to the developmental primordium (anlage) or even the homeotic identity, of the digits to which they are attached. Those results agree with previous studies focused on other tetrapod groups with non-pentadactyly as a wild-type phenotype, such as lizards, crocodiles, frogs, salamanders, and chickens, as well as humans with birth defects, and are therefore a new step towards the resolution of the old question about the specific associations between limb bones and muscles. Supported by CGL2014-52611-C2-2-P and PNPD/CAPES Brazil.

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