Transcript (ATF 4) in Zebrafish: Implications for Coffin

Expression Analysis of Activating Transcription Factor 4 (ATF 4)
in Zebrafish: Implications for Coffin-Lowry Syndrome
Jonathan Coale
Department of Biological Sciences York College of Pennsylvania
http://www.unsolvedmysteries.oregonstate.edu/embryo
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=cls&rendertype=figure&id=cls.F1
Introduction
- Coffin Lowry Syndrome (CLS) is a genetic disorder
that causes skeletal abnormalities due to a mutation
in the RSK2 (ribosomal S6 kinase) gene.
- Some CLS cases are caused by the lack of
phosphorylation of ATF4 (activating transcription
factor 4)by RSK2.
-ATF 4 is known for a variety of functions but it
mostly known for its role in bone development.
- ATF 4 has the ability to form homo/heterodimers
using its domains.
- Activated ATF 4 binds to its target gene, Osteocalcin
(Osn), which allows transcription of osteocalcin which
is essential for normal mineralization of bone.
Methods
Whole Adult ZF
Amino Acid Sequence
Selected Tissues
2,3, and 6 day
Old ZF Embryos
Extract RNA
Reverse Transcription
PCR
Red = βTrCP Recognition Site
Blue = Basic Domain
Green = Leucine Zipper
cDNA
PCR w/atf4
primers
5’
ATG (Start Codon)
Target Sequence (1019bp)
Forward Primer
3’
-In this study, zebrafish will be used to study the
function of ATF4 in development.
- The use of morpholino oligos in zebrafish could
show similar morphological differences to human
cases of CLS.
-Advantages of zebrafish ; translucent embryos, quick
rate of development, large quantity of offspring,
known genome, and ability to perform in situ
hybridization and morpholino knockdown
techniques.
TAG (Stop Codon)
3’
Reverse Primer
5’
Results
-Expression of ATF4 has been found to occur in
various adult tissues and 3 day old embryos.
-Key domains of ATF4 are conserved in zebrafish
when compared to human amino acid
sequence.
Future Research
-Finding where ATF4 is expressed in an embryo
via in situ hybridization.
-Observe morphological changes by introducing
morpholino oligos into embryos which examine
the effects of knocking down a gene.
Objectives
Literature Cited
-Is ATF4 expressed in adult zebrafish?
-What tissues show ATF4 expression in zebrafish?
-Is ATF4 expressed in embryonic zebrafish?
Conclusion
Figure 1. A 0.8% agarose gel of six cDNA tissue samples with ATF4
primers. In well one, 5ul of 500bp ladder was used and 10ul of
500bp ladder was used in well eight. (A=Adult, B=Brain, E=Eye,
G=Gut, H=Heart, M=Muscle)
Figure 2. A 1 % agarose gel ran at 240 volts. The numbers at the
top of each lane (2,3, and 6) represent the maturity of the embryo
(in days) when RNA was reverse transcribed to obtain cDNA
samples. The three day old embryo was the only sample with a
band at 1000bp meaning ATF 4 shows expression in a 3 day old
embryo.
-Yang X., Matsuda K., Bialek P., Jacquot S., Masuoka, C., Schinke T., Li L., (…)Karsenty G.
2004. ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology:
Implication for Coffin-Lowry syndrome. Cell, 117 (3), pp. 387-398.
-Ameri, K., Harris, A. 2008. Activating transcription factor 4. Science Direct. 40:14-21.
Acknowledgements: Special thanks to Dr. Wendy Boehmler for her guidance and patience.