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Session Schedule & Abstracts

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Friday 1st July, 2016

Evo-Devo - Evolution of Developmental Processes 1

Room: Salon G   2:30 pm–4:00 pm

Moderator(s): Debiais-Thibaud M, Rayfield EJ
EVD1-1  2:30 pm  The evolution of collagen and SPARC secretion during tooth development in vertebrates. Debiais-Thibaud M*, Montpellier University, France; Enault S, Montpellier University, France; Munoz D, University of Concepción, Chili; Ventéo S, INSERM U1051, Montpellier, France; Sire JY, CNRS UMR7138, Paris, France; Marcellini S, University of Concepción, Chili
Abstract: Vertebrate skeletal tissues develop from the activity of specific cells, all able to secrete a collagenous extra-cellular matrix which may or may not calcify. In this work, we describe the expression patterns of the major fibrillary collagen genes and genes of the Secreted Protein, Acidic, Cysteine-Rich (SPARC) family in two chondrichthyan species and one tetrapod where the SPARC family has not (chondrichthyans) or poorly (Xenopus) expanded into Secretory Calcium-binding PhosphoProtein (SCPP) duplicates. We show expression of all these genes in the mesenchymal compartment (odontoblasts) of teeth and the placoid scales of the catshark, except for the expression of SPARCL1 in the inner epithelium of chondrichthyan teeth and transient faint expression of SPARC in the Xenopus inner dental epithelium. Our results involve a restrained SPARCL1 expression in the epithelial layer of calcifying teeth (maturation stage) in chondrichthyans and no expression of collagen genes by ameloblasts. This result questions the cellular origin of chondrichthyan enameloid and its homology to enamel/enameloid found in osteichthyans. In contrast, a strongly conserved feature of odontoblasts in jawed vertebrate is therefore the co-expression of major fibrillar collagen genes and the SPARC gene. This observation calls for a putative gene regulatory network involved in extracellular matrix calcification that could also be shared between odontoblasts and osteoblasts of osteichthyans. These results therefore lead to various scenarios for the evolution of SPARC/SCPP genes in the gene regulatory networks involved in ameloblast and odontoblast function.

EVD1-2  2:45 pm  Reduction in tooth site regeneration underlies morphological novelty during pufferfish dental regeneration. Thiery AP, University of Sheffield; Fraser GJ*, University of Sheffield
Abstract: Consisting of approximately 25,000 species, morphological diversity within teleosts is rife. However, few morphological adaptations can rival the unique pufferfish ‘beak’, composed of multiple generations of parasymphyseal replacement teeth. In pufferfish, the transition between the first and second dental generation coincides with the emergence of this novel beaked morphology. As Sox2 has been identified as an important factor in maintaining dental regenerative competency in oral epithelium, we localised its expression in pufferfish. SOX2 is abundant throughout the oral epithelium but in conjunction with DiI cell labelling we highlight the labial oral epithelium as a putative dental stem cell site. We implicate canonical Wnt signalling in the activation of these dental progenitor cells and localise an odontogenic activation site to the base of the oral epithelium, labial to the first generation teeth. Despite the unusual morphology, we identified high levels of developmental conservation between pufferfish dental replacement and other vertebrate models studied. This study identifies a loss of dental replacement at all but four tooth sites as a fundamental driver of this morphological change. This elucidates how highly derived yet closely related teleosts have evolved extreme morphological novelty through modifications in dental number during rounds of dental regeneration.

EVD1-3  3:00 pm  Tammar wallaby Macropus eugenii (Macropodidae) as a model for tooth evolution, development, and replacement in mammals. Nasrullah Q*, Monash University; Renfree M, The University of Melbourne; Evans AR, Monash University
Abstract: Unlike their reptile-like ancestors, modern mammals replace their teeth only once (diphyodonty) or never (monophyodonty). Within mammals, eutherians and metatherians differ in the number of teeth in several of the tooth classes (incisor, canine, premolar and molar) and the mode of replacement. This study aims to resolve dental homology between eutherian and metatherian mammals using the Tammar wallaby Macropus eugenii as an opportune model for studying mammalian odontogenesis. Macropus eugenii has tooth replacement, four tooth classes, and – unusually among mammals – molar progression, but only preliminary investigations in the 1960s and 1980s have been carried out on its dental development. To provide a more comprehensive documentation of the spatio-temporal pattern of tooth development, we stained heads of pouch young aged between 0-135 days in 10% Lugol’s Iodine (I2KI), then microCT scanned using a Zeiss Xradia 520Versa and the micro-CT Imaging and Medical Beamline at the Australian Synchrotron. These were reconstructed and segmented in Avizo, generating 3D models. Our results reveal the position and orientation of developing tooth structures including: primary and secondary dental lamina; initiating tooth germs; major stages of tooth development (bud, bell and cap); and mineralised tissues. We tracked the overlapping development of two generations of teeth when present, and observed that deciduous incisors and canines were vestigial and cease development before eruption. The ‘stain and scan’ technique proved both time and cost effective in producing complex 3D models of the entire dentition at each stage with tissue-level resolution. Using these new models, we characterise tooth replacement in this marsupial by pinpointing the developmental origins of primary and secondary tooth generations, allowing us to clarify metatherian-eutherian dental homologies.

EVD1-4  3:15 pm  Developmental mechanism and genetic basis of the unique morphological characters of non-model organisms: investigation in bear molars as an example. Asahara M*, Mie University; Kishida T, Kyoto University
Abstract: The developmental mechanism and genetic basis of the unique morphology of non-model organisms and fossil taxa are interesting topics in biology; however, their investigation is difficult. Recently, a developmental model, the inhibitory cascade model, was proposed in a developmental study. The model explains the relative size of lower molars in mammals by a proportion of inhibition and activation molecules affecting the molar germ. According to the model, molar size decreases, is uniform, or increases along the molar row (M1 > M2 > M3, M1 = M2 = M3, or M1 < M2 < M3). Most mammals morphologically fit this model; accordingly, the model explains the variation in molar size in mammals. However, in bears, the second molar is the largest (M1 < M2 > M3); bears were considered an exception to the model, and the cause is unclear. Here, we used a combination of genetically modified mice, and morphological and molecular evolutionary analyses to reveal the cause of the unique molar in bears. We found a unique pattern of variation in relative molar sizes in the order Carnivora, i.e., lower slope of the regression of M3/M1 on M2/M1 than that in the original model. The molar pattern of bears appears to be an extension of this line. We also examined mice hetero-deficient for BMP7, a gene encoding an antagonist of a molecule involved in the model. Their molar morphology resembles the trend of variation in the Carnivora. Molecular evolutionary analysis revealed natural selection of the gene, especially of the domain binding its antagonist, in the ancestral lineage of bears. We conclude that variation in expression or affinity of low diffusible signaling molecules such as BMP7 can affect mesial molars more than distal molars, and it explains the variation in the Carnivora, including the unique phenotype of bears. Our method of combined analysis can be an example of investigating the unique phenotypes of non-model organisms.

EVD1-5  3:30 pm  Morphology and function of the toothrow in a rodent knockout model and implications for mammalian tooth evolution . Zurowski C*, University of Calgary ; Jamniczky H, University of Calgary; Graf D, University of Alberta ; Theodor J, University of Calgary
Abstract: Tooth morphology is the result of many complex tissue interactions within the developing tooth. Differences in cusp shape, size and orientation provide evidence of phylogeny, as well as alterations in feeding strategy and amount of intraoral processing. Many families of regulatory genes play key roles in the resultant morphology of the tooth. Determining and quantifying the effect of these regulatory genes on the morphology and function of mammalian dentition has implications for understanding the underlying mechanisms that drove the degree of dental diversity that we see in both extinct and extant mammals. We tested the hypothesis that changes in regulatory gene expression can lead to changes in morphology and function of the toothrow using a neural crest specific knockout of the first coding region, exon 1, of bone morphogenetic protein 7 (BMP7). These BMP7 mutants have distinctive craniofacial morphology, which includes noticeably altered tooth morphology. Mutant molars have extra cusps, mostly on the first upper and lower molars, along with differences in cusp morphology. The cusps on mutant molars are shorter and blunter than the control cusps and the orientation of the cusps in relation to other cusps differs. To quantify differences in morphology, a landmark set was developed and geometric morphometric methods were applied to 3D models of the right upper and lower toothrows. Significant morphological differences between the control and BMP7 mutant mice were found for both the upper and lower toothrows. Additionally, mutant and control mice were found to have different wear facets, indicating that along with a change in morphology, there was also a change in function. This research shows that changes in the expression of BMP7 can lead to changes in the morphology and function of the toothrow and suggests that BMP7 could have played a role in structuring the amount of dental diversity that we see in extinct and extant mammals.

EVD1-6  3:45 pm  The influence of mechanical loading on jaw joint morphology during development. Rayfield EJ*, University of Bristol; Brunt LH, University of Bristol; Bright JA, University of Sheffield; Roddy KA, University of Bristol; Hammond CL, University of Bristol
Abstract: It is accepted that mechanical loading via muscle activity is required for normal skeletal development, particularly at joint contact surfaces. Despite this, little is known about the effect of disrupted mechanical loading on craniofacial skeletal development. Using zebrafish as a model system, genetic and pharmacological studies have demonstrated that mechanical loading is required for accurate joint morphogenesis. Using anaesthetised and genetically manipulated fish, here we show that removal of muscle activity during zebrafish development results in a reorganisation of jaw joint (Meckel’s cartilage and palatoquadrate) shape from a ball and socket articulation to a flattened and overgrown joint surface. We find that control fish begin to voluntarily open and close their mouths at days 4 to 5 and that muscle activity at this time is crucial for normal joint formation. Removal of muscle loads at days 4 to 5 results in abnormal joint formation, whereas removal of loads earlier during development has little effect on joint formation and most fish develop normally once muscle activity is renewed. After recording the number of adductor muscle fibres, fibre area and estimated muscle force, we use finite element modelling to reconstruct strains within the developing cartilages. Abnormal joint morphology modifies strains within the developing jaw cartilages, and, when muscle-induced strain is removed, cells on the medial side of the joint change their orientation. Our results suggest that muscle-induced strain regulates cell orientation at the developing joint and that biomechanical loading via adductor muscle contraction plays a key role in normal zebrafish jaw joint formation.

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