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A Line of Zebrafish with Development of Abnormal Spinal Curvature 1HG.Tomasiewicz, Ph.D; 2J. Thometz, M.D; 2XC. Liu, MD, Ph.D; 2C. Tassone, M.D; 3P. North, M.D 1NIEHS Children’s Environmental Health Sciences Core Center, University of Wisconsin-Milwaukee 2Dept. of Orthopaedic Surgery, 3Dept.of Pathology, Children’s Hospital of Wisconsin, Medical College of Wisconsin, Milwaukee, WI Introduction • Difficult to create animal models which mimic human scoliosis • The chicken is a commonly used model, followed by the rabbit & rat • Drastic surgical or systemic interventions required to create a curve * Pinealectomy * Tethering systems * Environmental toxins Beuerlein M, et al. The critical stage of pinealectomy surgery after which scoliosis is produced in young chickens. Spine 2001. Carpintero P, etc. Scoliosis induced by asymmetric lordosis and rotation: an experimental study. Spine 1997 Introduction • • • The lack of a good animal model system has hindered studying the etiology of idiopathic scoliosis Difficult to recreate 3D deformity seen in humans in animals which are not upright Several fish species appear to exhibit spinal curvatures (Zebrafish, Salmon, etc) Objectives • To determine if the affected Zebrafish with spinal deformities resemble the pathology observed in scoliosis Opportunity • Collaboration with NIEHS • Zebrafish genome completely mapped Methods • Founder fish with spinal curvatures were outcrossed with a wild type Zebrafish line (AB) and the resulting siblings (F1 generation) were crossed • The offspring (F2 generation) examined for signs of spinal curvature beginning at 14 days post fertilization (dpf) Faxitron Image, Video, Histology, Polymerase chain reaction (PCR) Methods • Microsporidia infections are known to cause spinal deformities in Zebrafish; need to exclude this as etiology • A and B: (+) PCR Microsporidia infected fish as seen in sections of Zebrafish spinal cord • C: Fish with scoliosis • D: (-) PCR Results • Range from 18° to 40° SD Mutant Wild Type Ferguson method for measurement; location of curve-distal to dorsal fins High speed, high resolution camera Results Histology Vertebral Body Vertebral Endplate SD Mutant Wild Type Shortened Deformed Widened, Distorted Endplate Results • 212 individuals in the F2 generation - 28, or 13.2%, had spinal deformities at 21 dpf. • No spinal deformities in the F1 generation fish and similar age wild type fish, indicating the observed spinal deformities were due to a recessive mutation(s). Conclusion • “Scoliosis” in these Zebrafish results from mutation(s) in the Zebrafish genome • Offspring exhibit a predictable frequency of scoliosis which can be used to study the etiology and progression of scoliosis. Future • Continue to develop colonies of Zebrafish with Scoliosis • Evaluate candidate genes from Zebrafish research • Create Knockout fish • This line of Zebrafish could help identify the etiology of Scoliosis Acknowledgement UWM’s NIEHS Children’s Environmental Health Sciences Core Center Orthopaedic Surgery Department, MCW