Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.

BACKGROUND The relative effects of skin growth and stretch during tissue expansion have not been studied. The authors use novel analytic techniques that allow calculation of these factors at any point of a skin patch. OBJECTIVE The authors sought to determine how stretch and growth change with different expansion rates and to correlate these values with histologic and cellular changes in skin. MATERIALS AND METHODS Two minipigs were implanted with a total of 5 tissue expanders under tattooed skin grids. One pig was expanded over 35 days and the second over 15 days. Isogeometric analysis allowed calculation of growth and stretch. Expanders with similar total deformation were compared between protocols. Regression analysis determined predictive effects of stretch and growth on histologic data from the second animal. RESULTS Deformation was more attributable to stretch in rapid than in slow expansion (1.40 vs1.12, p < .001). Growth was higher in slow expansion than in rapid (1.52 vs 1.07, p < .001). Both growth and stretch predicted epidermal thickness, dermal thinning, and keratinocyte proliferation. Growth predicted vascularity. CONCLUSION Isogeometric analysis allows determination of precise surface area changes for correlation to microscopic-level data. Using the model, the authors identified that skin deformation in rapid expansion is more attributable to stretch.

PurPose: Mutations in TWIST and FGFR genes have been implicated in the pathogenesis of craniosynostosis in developing humans. However, these genes also play a significant role in mesenchymal cell proliferation and differentiation throughout the developing organism, making it difficult to study isolated mutations in the craniofacial skeletal tissues. The present study was undertaken to (1) determine if genes implicated in the development of human craniosynostosis could be localized in the zebrafish cranial vault; and (2) if so, determine if we can successfully manipulate expression of these genes locally, thereby avoiding the systemic implications of a germline mutation.Methods: Digoxigenin (DIG)-labeled RNA antisense probes were designed to bind the genes of interest for in-situ hybridization (twist1a, twist1b, twist2, twist3). Wild-type fish calvaria were dissected after fixation with paraformaldehyde (PFA) solution for 24 hours. Half of the skulls underwent an additional bleaching protocol utilizing hydrogen peroxide and potassium hydroxide to remove pigmentation prior to in-situ hybridization. Fish calvaria were imaged using standard light microscopy. The transgenic zebrafish line Tg(dnFGFR1:EGFP) was used to develop a protocol for localized heat shock using a modified soldering iron based on previous work (Hardy, et al. 2007). Tricaine-anesthetized fish were immobilized, and heat shock was applied to the cranial sutures using a fine-tipped soldering iron at a constant energy of 28.5V for three-minute intervals. Gene expression was detected by GFP reporter expression using confocal microscopy. results: RNA in-situ hybridization revealed that twist1a was not expressed in the sutures of the developing zebrafish; however, twist1b, twist2, and twist3 all localized to different suture regions in the developing calvaria. The localized heat shock approach successfully induced expression of the dominant-negative Fgfr1 mutant protein in the coronal sutures of Tg(dnFGFR1:EGFP) juvenile fish, thus establishing this method for future manipulations.ConClusions: TWIST genes are highly conserved across species, and encode regulatory proteins that are essential to embryonic development. Specifically, Twist mutations have been implicated in human craniosynostosis. Here, we characterize the differential expression patterns of the zebrafish TWIST gene homologs--twist1a, twist1b, twist2, and twist3--in the developing cranial sutures. Moreover, through the application of localized heat shock, we have been able to locally induce expression of an engineered transgene with downstream GFP reporters. We believe we can expand upon this model of isolated gene expression in specific tissues of interest. This technology can allow for the regulation of abnormal cranial suture development without influencing the broader activity of target genes in the developing organism.