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Title: ALTERNATE PATCHED SPLICE FORMS ARE EXPRESSED IN A TISSUE SPECIFIC MANNER DURING EARLY EMBRYONIC DEVELOPMENT

Author
item HARRIS, LESLIE - BAYLOR COLLEGE MED
item BUKOWSKI, JOHN - BAYLOR COLLEGE MED
item Karpen, Heidi

Submitted to: Pediatric Research
Publication Type: Abstract Only
Publication Acceptance Date: 4/1/2006
Publication Date: 5/1/2006
Citation: Harris, L.L., Bukowski, J.T., Karpen, H.E. 2006. Alternate patched splice forms are expressed in a tissue specific manner during early embryonic development [abstract]. Pediatric Research. E-PAS2006. 59:2876.318.

Interpretive Summary:

Technical Abstract: BACKGROUND: The Hedgehog (Hh) pathway is critical for embryonic patterning of nearly every organ system in the developing fetus and is highly conserved across phylogeny. We have previously characterized three alternate splice forms of the Ptc gene, including a novel Exon 1C isoform in the mouse, but their significance in embryonic development remains unclear. OBJECTIVE: Show that the timing and level of alternate Ptc splice form expression provides precise, tissue-specific regulation of the Hh pathway during critical points in early embryonic development. DESIGN/METHODS: Relative expression of Ptc alternate splice forms during early development was assessed by QRT-PCR of RNA from E7, E11, E15, and E17 whole mouse embryos. This experiment was then repeated on mouse embryos obtained at 24-hour intervals from E7.5 to E12.5. Initial studies of differential tissue expression were performed by QRT-PCR of adult mouse tissues. Embryonic tissue specific expression was assessed by in-situ hybridization (ISH) of both whole mount and slide sections from E7.5-E12.5 embryos. RESULTS: QRT-PCR of whole mouse embryo RNA from E7, E11, E15, and E17 revealed a 7- to 8-fold rise in both the Exon 1B and Exon 1C isoforms between E7 and E11. Exon 1 exhibited an earlier peak at E7, suggesting different mechanisms or target tissues. Analysis of earlier embryonic time intervals showed the greatest rise in Ptc 1B and 1C expression between E7.5 and E8.5, corresponding to the initiation of organogenesis in the mouse embryo. QRT-PCR of individual tissues showed the highest level of Exon 1B and 1C expression in the brain, which is consistent with the brain being the most Hh dependent organ. Exon 1 expression was high in the heart, liver, and kidney. ISH revealed differential expression and localization of each exon at different time points, with high levels of 1B expressed in the developing brain and limbs. CONCLUSIONS: This study has shown that there is significant variation in the expression of the individual Ptc isoforms by tissue and gestational stage. This differential tissue expression suggests that these alternate splice forms likely provide a more refined level of control in a critical pathway during embryonic development. Differential expression is likely driven by specific elements in the exon specific promoter regions.