cAMP Response Element Binding Protein Is Required for Differentiation of Respiratory Epithelium during Murine Development A. Daniel Bird 1 *, Sharon J. Flecknoe 2 , Kheng H. Tan 1 , P. Fredrik Olsson 1 , Nisha Antony 1 , Theo Mantamadiotis 3 , Stuart B. Hooper 2 , Timothy J. Cole 1 1 Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, Australia, 2 The Ritchie Centre, Monash Institute of Medical Research, Clayton, Victoria, Australia, 3 Department of Medicine, University of Patras, Patras, Greece Abstract The cAMP response element binding protein 1 (Creb1) transcription factor regulates cellular gene expression in response to elevated levels of intracellular cAMP. Creb1 2/2 fetal mice are phenotypically smaller than wildtype littermates, predominantly die in utero and do not survive after birth due to respiratory failure. We have further investigated the respiratory defect of Creb1 2/2 fetal mice during development. Lungs of Creb1 2/2 fetal mice were pale in colour and smaller than wildtype controls in proportion to their reduced body size. Creb1 2/2 lungs also did not mature morphologically beyond E16.5 with little or no expansion of airway luminal spaces, a phenotype also observed with the Creb1 2/2 lung on a Crem 2/2 genetic background. Creb1 was highly expressed throughout the lung at all stages examined, however activation of Creb1 was detected primarily in distal lung epithelium. Cell differentiation of E17.5 Creb1 2/2 lung distal epithelium was analysed by electron microscopy and showed markedly reduced numbers of type-I and type-II alveolar epithelial cells. Furthermore, immunomarkers for specific lineages of proximal epithelium including ciliated, non-ciliated (Clara), and neuroendocrine cells showed delayed onset of expression in the Creb1 2/2 lung. Finally, gene expression analyses of the E17.5 Creb1 2/2 lung using whole genome microarray and qPCR collectively identified respiratory marker gene profiles and provide potential novel Creb1-regulated genes. Together, these results demonstrate a crucial role for Creb1 activity for the development and differentiation of the conducting and distal lung epithelium. Citation: Bird AD, Flecknoe SJ, Tan KH, Olsson PF, Antony N, et al. (2011) cAMP Response Element Binding Protein Is Required for Differentiation of Respiratory Epithelium during Murine Development. PLoS ONE 6(3): e17843. doi:10.1371/journal.pone.0017843 Editor: Saverio Bellusci, Children’s Hospital Los Angeles, United States of America Received November 29, 2010; Accepted February 12, 2011; Published March 8, 2011 Copyright: ß 2011 Bird et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a program grant of the National Health and Medical Research Council of Australia (ID no. 384100). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: daniel.bird@monash.edu Introduction Survival at birth is critically dependent upon the ability of the lung to immediately take over the role of gas exchange, which in turn, is dependent upon the development of a mature respiratory system during the fetal period. The continuously branching terminal buds of the developing airways contain a population of multipotent epithelial stem or progenitor cells that give rise to the major cell types of the mature airway epithelium [1,2]. Neuroepithelial, ciliated, and finally non-ciliated secretory Clara cells differentiate within the airway buds that undergo branching morphogenesis during the pseudoglandular stage (,E10.5–E16.5), while more-distally located alveolar epithelial cells (AECs) begin to differentiate later during the canalicular stage (E16.5–E17.5). Correct differentiation into these cell types, particularly those populating the distal epithelium, is essential to ensure sufficient levels of pulmonary surfactant and surface area for gas exchange, which together are required for survival at birth. The molecular mechanisms regulating differentiation of the lung epithelium into its specialised cell types during development are only partially understood, however it is clear that specific nuclear transcription factors, often acting in complex regulatory networks, play a critical role in mediating this process [3]. In this report we have investigated the role of Creb1 (cyclic adenosine 39,59-monophos- phate (cAMP) response element binding protein), a downstream transcriptional mediator for a range of systemic signalling factors, in respiratory epithelial differentiation. Creb1 is a member of the Creb/Atf subfamily of cAMP- responsive basic region-leucine zipper (bZIP) transcription factors which include activating transcription factor 1 (Atf1) and the cAMP response element modulatory protein (Crem) [4]. A large range of signalling factors including steroid hormones, peptide hormones, growth factors and other cytokines can induce activation of Creb1, usually via stimulation of pathways which increase intracellular levels of cAMP. Elevated levels of cAMP activate and release catalytic subunits of protein kinase A (PKA) which phosphorylate Creb1 primarily at Ser133 [5,6,7]. Once phosphorylated, Creb1 can bind as homo- or heterodimers with other Creb/Atf subfamily members to cAMP response elements or CREs (‘TGACGTCA’) in promoter regions and transactivate specific target genes [4]. Gene targeted mutations of the Creb/Atf1 subfamily in mice have provided important insight into the biological function of these factors during development. Mice lacking Atf1 and Crem PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e17843