Download A Line of Zebrafish with Development of Abnormal Spinal Curvature

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