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NPAS1 regulates branching morphogenesis in embryonic lung.

ABSTRACT: 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 alpha-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: beta-ig-h3, claudin-11, and myocardin. Npas1 can regulate myofibroblast distribution, branching morphogenesis, and neuroendocrine cell differentiation in murine embryonic lung.

SUBMITTER: Levesque BM

PROVIDER: S-EPMC1899329 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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NPAS1 regulates branching morphogenesis in embryonic lung.

Levesque Bernadette M BM Zhou Shutang S Shan Lin L Johnston Pamela P Kong Yanping Y Degan Simone S Sunday Mary E ME

American journal of respiratory cell and molecular biology 20061116 4

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 alpha-smooth muscle actin demonstrated myofibroblasts, and protein gene product (PGP)9.5 identified neuroendocrine cells. Branching morphogenesis of ...[more]

PMID: 17110583

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Project description:Prostatic branching morphogenesis is an intricate event requiring precise temporal and spatial integration of numerous hormonal and growth factor-regulated inputs, yet relatively little is known about the downstream signaling pathways that orchestrate this process. In this study, we use a novel mesenchyme-free embryonic prostate culture system, newly available mTOR inhibitors and a conditional PTEN loss-of-function model to investigate the role of the interconnected PI3K and mTOR signaling pathways in prostatic organogenesis. We demonstrate that PI3K levels and PI3K/mTOR activity are robustly induced by androgen during murine prostatic development and that PI3K/mTOR signaling is necessary for prostatic epithelial bud invasion of surrounding mesenchyme. To elucidate the cellular mechanism by which PI3K/mTOR signaling regulates prostatic branching, we show that PI3K/mTOR inhibition does not significantly alter epithelial proliferation or apoptosis, but rather decreases the efficiency and speed with which the developing prostatic epithelial cells migrate. Using mTOR kinase inhibitors to tease out the independent effects of mTOR signaling downstream of PI3K, we find that simultaneous inhibition of mTORC1 and mTORC2 activity attenuates prostatic branching and is sufficient to phenocopy combined PI3K/mTOR inhibition. Surprisingly, however, mTORC1 inhibition alone has the reverse effect, increasing the number and length of prostatic branches. Finally, simultaneous activation of PI3K and downstream mTORC1/C2 via epithelial PTEN loss-of-function also results in decreased budding reversible by mTORC1 inhibition, suggesting that the effect of mTORC1 on branching is not primarily mediated by negative feedback on PI3K/mTORC2 signaling. Taken together, our data point to an important role for PI3K/mTOR signaling in prostatic epithelial invasion and migration and implicates the balance of PI3K and downstream mTORC1/C2 activity as a critical regulator of prostatic epithelial morphogenesis.

Project description:An elaborate system of ducts collects urine from all nephrons, and this structure is known as the urinary collecting system (UCS). This study focused on how the UCS is formed during human embryogenesis. Fifty human embryos between the Carnegie stage (CS) 14 and CS23 were selected from the Kyoto Collection at the Congenital Anomaly Research Center of Kyoto University, Japan. Metanephroses, including the UCS, were segmented on serial digital virtual histological sections. Three-dimensional images were computationally reconstructed for morphological and quantitative analyses. A CS timeline was plotted. It consisted of the 3-D structural morphogenesis of UCS and quantification of the total amount of end-branching, average and maximum numbers of generations, deviation in the metanephros, differentiation of the urothelial epithelium in the renal pelvis, and timing of the rapid expansion of the renal pelvis. The first UCS branching generation occurred by CS16. The average branching generation reached a maximum of 8.74 ± 1.60 and was already the twelfth in CS23. The total end-branching number squared between the start and the end of the embryonic period. UCS would reach the fifteenth branching generation soon after CS23. The number of nephrons per UCS end-branch was low (0.21 ± 0.14 at CS19, 1.34 ± 0.49 at CS23), indicating that the bifid branching occurred rapidly and that the formation of nephrons followed after. The renal pelvis expanded mainly in CS23, which was earlier than that reported in a previous study. The number of nephrons connected to the UCS in the expanded group (246.0 ± 13.2) was significantly larger than that of the pre-expanded group (130.8 ± 80.1) (P < 0.05). The urothelial epithelium differentiated from the zeroth to the third generations at CS23. Differentiation may have continued up until the tenth generation to allow for renal pelvis expansion. The branching speed was not uniform. There were significantly more branching generations in the polar- than in the interpolar regions (P < 0.05). Branching speed reflects the growth orientation required to form the metanephros. Further study will be necessary to understand the renal pelvis expansion mechanism in CS23. Our CS-based timeline enabled us to map UCS formation and predict functional renal capacity after differentiation and growth.

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NPAS1 regulates branching morphogenesis in embryonic lung.

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NPAS1 regulates branching morphogenesis in embryonic lung.

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