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ARS Home » Plains Area » Houston, Texas » Children's Nutrition Research Center » Research » Publications at this Location » Publication #201903
Title: Nkx3.2 AND HEDGEHOG PATHWAY INTERSECT TO PATTERN AXIAL SKELETON

Author
item STANFEL, MONIQUE - BAYLOR COLLEGE MED
item BUKOWSKI, JOHN - BAYLOR COLLEGE MED
item SCHWARTZ, ROBERT - TAMU IBT-HOUSTON,TX
item ZIMMER, WARREN - TAMU HSC-COLLEGE STA.TX
item Karpen, Heidi

Submitted to: Pediatric Research
Publication Type: Abstract Only
Publication Acceptance Date: 4/1/2006
Publication Date: 5/1/2006
Citation: Stanfel, M.N., Bukowski, J.T., Schwartz, R.J., Zimmer, W.E., Karpen, H.E. 2006. Nkx3.2 and hedgehog pathway intersect to pattern axial skeleton [abstract]. Pediatric Research. E-PAS2006. 59:2876.319.

Interpretive Summary:

Technical Abstract: BACKGROUND: NK homeobox family members are tissue-specific transcription factors important for regulation of developmental genes. Nkx3.2 is one of the earliest markers of sclerotome and gut mesoderm. Targeted disruption of Nkx3.2 expression in mice produced severe developmental defects, including malformations of the axial skeleton, skull, spleen, and stomach. The Hedgehog (Hh) pathway is also critical for embryonic patterning at coincident points in development. Heterozygous disruption of the Hedgehog receptor Patched1 (Ptc1), resulted in a propensity for cancers and multiple developmental anomalies, including skeletal anomalies. OBJECTIVE: Nkx3.2 participates in epithelial-mesenchyme signaling by regulating expression of other factors involved in embryonic patterning, such as Nkx2.5 and Hh pathway members. DESIGN/METHODS: Microarray (Amersham) and RT-PCR assays were performed using E18.5 mouse embryo total RNA. Raw data was analyzed using Microsoft Excel, normalized on a log2 scale, and filtered. Gene ontology analysis was performed using GeneSpring 7.2. NIH3T3 cells were stimulated with soluble Shh-N, and QRT-PCR analysis was performed to assess Nkx3.2 expression. A Gli reporter construct was transfected in parallel to verify Hh pathway activation. Whole mount in situ hybridization (ISH) was performed on Shh mutant embryos using cRNA probes for Nkx3.2 synthesized using a Roche DIG-labeling kit. RESULTS: Microarray analysis and semi-quantitative RT-PCR results indicated Ptc1 expression decreased in cervical and pyloric sphincter regions of Nkx3.2-null E18.5 mouse embryos. QRT-PCR analysis of RNA from NIH3T3 cells stimulated with soluble Shh-N show increased Nkx3.2 expression. ISH shows Nkx3.2 expression is absent in the somites of Shh mutant embryos. In addition, multiple potential Nkx3.2 binding sites were identified in the Ptc1 promoter. CONCLUSIONS: These results indicate that Nkx3.2 expression was responsive to Shh signaling. Disruption of Nkx3.2 in vivo resulted in decreased Ptc1 expression in tissue from E18.5 mouse Nkx3.2 KO embryos. Our studies indicate that Nkx3.2 is an upstream modulator of Ptc1 expression. Nkx3.2 is likely involved in restricting Hh signaling and may play an important role in patterning of the axial skeleton.