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Identification of twist Expression Patterns and Localized Manipulation of fgfr1 Expression in Zebrafish Cranial Sutures
Michael S. Gart, MD1, Joanna P. Tomaszewski, MS2, Jolanta M. Topczewska, PhD2, Arun K. Gosain, MD3.
1Nowthwestern University Feinberg School of Medicine, Division of Plastic Surgery, Chicago, IL, USA, 2Nowthwestern University Feinberg School of Medicine, Department of Surgery, Chicago, IL, USA, 3Ann & Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, USA.
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.
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 (Figure 1).
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 (Figure 2).
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.
Figure 1: twist expression patterns in cranial sutures (A, twist1a; B, twist1b; C, twist2, bleached; D, twist3, bleached).
Figure 2: Confocal microscopy of zebrafish treated with localized heat shock, showing focal expression of transgene (GFP) (A, low magnification; B, 40x magnification).
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