This Article Full Text Full Text (PDF) Online Supplement All Versions of this Article: 2006-0314OCv1 36/4/427    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Levesque, B. M. Articles by Sunday, M. E. Search for Related Content PubMed PubMed Citation Articles by Levesque, B. M. Articles by Sunday, M. E.

NPAS1 Regulates Branching Morphogenesis in Embryonic Lung

Department of Pediatrics, and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, Massachusetts; and Department of Pathology, Duke University Medical Center, Durham, North Carolina

Correspondence and requests for reprints should be addressed to Mary E. Sunday, M.D., Ph.D., Department of Pathology, Duke University Medical Center, Research Drive, Carl Building, Room 0043, Durham, NC 27710. E-mail: mary.sunday{at}duke.edu

Drosophila trachealess (Trl), master regulator of tracheogenesis, has no known functional mammalian homolog. We hypothesized that genes similar to trachealess regulate lung development. Quantitative (Q)RT-PCR and immunostaining were used to determine spatial and temporal patterns of npas1 gene expression in developing murine lung. Immunostaining for -smooth muscle actin demonstrated myofibroblasts, and protein gene product (PGP)9.5 identified neuroendocrine cells. Branching morphogenesis of embryonic lung buds was analyzed in the presence of antisense or sense oligodeoxynucleotides (ODN). Microarray analyses were performed to screen for changes in gene expression in antisense-treated lungs. QRT-PCR was used to validate the altered expression of key genes identified on the microarrays. We demonstrate that npas1 is expressed in murine embryonic lung. npas1 mRNA peaks early at Embryonic Day (E)10.5–E11.5, then drops to low levels. Sequencing verifies the identity of npas1 transcripts in embryonic lung. NPAS1 immunostaining occurs in nuclei of parabronchial mesenchymal cells, especially at the tracheal bifurcation. Arnt, the murine homolog of Tango (the heterodimerization partner for Trl) is also expressed in developing lung but at constant levels. npas1- or arnt-antisense ODN inhibit lung branching morphogenesis, with altered myofibroblast development and increased pulmonary neuroendocrine cells. On microarrays, we identify > 50 known genes down-regulated by npas1-antisense, including multiple genes regulating cell migration and cell differentiation. QRT-PCR confirms significantly decreased expression of the neurogenic genes RBP-Jk and Tle, and three genes involved in muscle development: –ig-h3, claudin-11, and myocardin. Npas1 can regulate myofibroblast distribution, branching morphogenesis, and neuroendocrine cell differentiation in murine embryonic lung.

Key Words: branching morphogenesis • myofibroblasts • smooth muscle actin • cell migration • neuroendocrine cells

CLINICAL RELEVANCE This research identifies NPAS1 as a novel transcription factor regulating branching morphogenesis in murine lung buds. NPAS1 antisense oligos inhibit branching via a mechanism involving arrested myofibroblast migration and neuroendocrine cell hyperplasia.