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ARS Home » Plains Area » Houston, Texas » Children's Nutrition Research Center » Research » Publications at this Location » Publication #215073
Title: Nkx factors specifically regulate expression of Hedgehog receptor isoforms in early embryonic development

Author
item STANFEL, MONIQUE - IBT/TAMU HOUSTON,TX
item BUKOWSKI, JOHN - BAYLOR COLLEGE MED
item SCHWARTZ, ROBERT - IBT/TAMU HOUSTON,TX
item ZIMMER, WARREN - IBT/TAMU HOUSTON, TX
item KARPEN, HEIDI - BAYLOR COLLEGE MED

Submitted to: Pediatric Academic Society
Publication Type: Abstract Only
Publication Acceptance Date: 3/1/2007
Publication Date: 4/1/2007
Citation: Stanfel, M.N., Bukowski, J.T., Schwartz, R.J., Zimmer, W.E., Karpen, H.E. 2007. Nkx factors specifically regulate expression of Hedgehog receptor isoforms in early embryonic development [abstract]. Pediatric Academic Society. E-PAS2007:616301.7.

Interpretive Summary:

Technical Abstract: BACKGROUND: NK homeobox family members are tissue-specific transcription factors that regulate developmental genes. Homozygous disruption of Nkx3.2 produces severe developmental defects of the axial skeleton, skull, spleen, and stomach. Murine mutation of Nkx2.5 results in death at E9 with defects in cardiac development. The Hedgehog (Hh) pathway is also critical for embryonic patterning at coincident developmental points. Mutations the Hh receptor Patched (Ptc) result in multiple developmental defects and homozygous disruption is embryonic lethal. OBJECTIVE: Nkx 2.5 and Nkx3.2 direct key events in epithelial-mesenchyme signaling by interacting with and regulating the expression of Hh pathway members during critical points in embryonic development. DESIGN/METHODS: RT-PCR performed on E18.5 mouse embryo total RNA. NKEs/Gli sites mapped by rVISTA. Luciferase assays performed on C310T1/2 cells. NKEs mutated using site directed mutagenesis (SDM). Nkx 3.2 binding studies performed with ChIP. QRT-PCR performed on WT E7.5-E12.5 mouse tissues. RESULTS: RT-PCR of E18.5 Nkx3.2 KO mouse cervical tissue shows decreased Ptc1 expression. QRT-PCR of WT embryonic tissues shows co-expression of Nkx factors/Ptc isoforms. Using rVista analysis, luciferase assays and ChIP, we characterized multiple Nkx factor binding elements (NKE) in Ptc exon specific promoters. These NKE show differential response to Nkx factor stimulation in vitro. Nkx3.2 stimulates the 1C-Ptc promoter, and synergizes with Gli1, while Nkx2.5 stimulates the Exon1-Ptc promoter. Competition studies indicate that Nkx 3.2 and 2.5 compete for binding of the same NKE in the Exon1-Ptc promoter and may regulate Ptc tissue specific isoform switching. ChIP experiments show specific binding of Nkx 3.2 to the 1C-Ptc promoter. SDM of specific NKEs ablates Nkx factor stimulation of the Ptc promoters, showing these interactions are specific. CONCLUSIONS: Disruption of Nkx3.2 in vivo results in decreased Ptc1 expression in tissue from E18.5d mice. We have identified and characterized multiple NKEs in the individual Ptc1 promoter regions, which respond differentially to Nkx factor stimulation. Nkx2.5 and Nkx3.2 act as upstream modulators of Ptc1 expression and appear to control tissue specific Ptc isoform expression and isoform switching.