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Smad-interacting protein-1 (SIP1), which has been implicated in the development of Hirschsprung disease-mental retardation syndrome, is thought to be a transcriptional repressor of the BMP signaling pathway. The attenuation of the BMP pathway is responsible for induction of neural tissue in the developing embryo. Previous studies on SIP1 orthologues have shown that SIP1 may indeed be a player in the expression of early neural markers and in the differentiation of neural tissue. Thus, we hypothesized that a zebrafish SIP1 orthologue would be expressed in the neural precursors of the early embryo and may be involved in neural specification and differentiation. In this study we identified and subcloned two SIP1 orthologues in zebrafish. After subcloning the zebrafish orthologues, we generated antisense digoxigenin-labeled probes to analyze the expression of the two orthologues. We also generated and injected translation blocking morpholino antisense oligonucleotides to reduce the levels of SIP1 and further characterize its role in the development of the zebrafish embryo. We found that the two zebrafish orthologues had very different expression patterns in the early embryo, though both were localized throughout early embryogenesis to developing neural tissue. Morpholino injected embryos for both orthologues were found to have dorsal-ventral patterning defects. Further study of these zebrafish orthologues may provide insight into the role of SIP1 in neural differentiation and specifically in the development of Hirschsprung disease.
Hirschsprung disease is a genetic condition where there is a lack of neurons at the bottom portion of the large intestine, so that the gut is not able to undergo normal peristalsis and pass material through the gut properly. There is a range of severity associated with the disorder, but in the most severe cases, newborn infants start to bloat within a few days of birth since they cannot pass anything out of their system. As of now, the only solution is to surgically remove the affected portion of the gut. Our lab is interested in studying genes which may be responsible for this condition. We use the zebrafish as a model system because they produce large quantities of large, transparent embryos and are a good system for genetic studies.
Other labs have found that the gene SIP1 may be responsible for a more severe form of the disorder called Hirschsprung disease-mental retardation syndrome. The gene is thought to induce the formation of neural tissue in the embryo. Our lab is currently studying where the gene is expressed in normal embryos and using biological techniques to knock down the gene and see its effects. We use markers for different types of neural tissue to see if correct cell types can still form when SIP1 is not expressed in the embryo. Through this project, we hope to gain a better understanding of the role of SIP1 in the development of neural tissue and specifically in Hirschsprung disease.
RT-PCR, gel electrophoresis, RACE (Rapid Amplification of cDNA Ends) PCR, DNA extraction from gel, Ligating DNA into plasmids and subsequent transformation into competent cells, Restriction enzyme cuts , In situ hybridization
enteric nervous system, Hirschsprung diseas, neural crest, BMP signaling, dorsal-ventral patterning
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