Regulation of retinoic acid signaling during lung morphogenesis

Sarah Malpel 1 Cathy Mendelsohn 0 Wellington V. Cardoso ) 1 0 Columbia University, Department of Urology and Pathology , New York, NY, 10032 , USA 1 Pulmonary Center - Boston University School of Medicine , Boston, MA 02118 , USA SUMMARY Little is known about how retinoic acid (RA) synthesis, utilization and metabolism are regulated in the embryonic lung and how these activities relate to lung pattern formation. Here we report that early lung bud formation and subsequent branching morphogenesis are characterized by distinct stages of RA signaling. At the onset of lung development RA signaling is ubiquitously activated in primary buds, as shown by expression of the major RA-synthesizing enzyme, RALDH-2 and activation of a RARE-lacZ transgene. Nevertheless, further airway branching appears to require downregulation of RA pathways by decreased synthesis, increased RA degradation in the epithelium via P450RAI-mediated metabolism, and inhibition of RA signaling in the mesenchyme by COUPTF-II expression. These mechanisms controlling local RA signaling may be critical for normal branching, since we show that manipulating RA Although several studies have shown that retinoids are important for proper lung morphogenesis and for differentiation of the respiratory epithelium, little is known about the ontogeny of retinoic acid (RA) signaling in the lung and the role of RA in lung pattern formation. Maternal administration of teratogenic doses of retinoids as well as retinoid deprivation cause marked lung dysmorphogenesis in embryos (Wilson et al., 1953; Shenefelt et al., 1972; Dickman et al, 1997). Dramatic abnormalities that resemble those observed in retinoid-deficient conditions are found in compound RA receptor (RAR) null mutant mice. However, the distribution of these malformations in the lung and other organs does not correlate well with the sites where RARs are normally expressed (Mendelsohn et al., 1994). While this discrepancy could result from disruption of RA-dependent events outside the lung, it also indicates that, besides distribution of retinoid receptors, other factors such as ligand bioavailability and the presence of RA activators or repressors also control RA signaling in the lung. RA is generated by a series of oxidative reactions that convert retinol to retinaldehyde and ultimately to the active form retinoic acid (Zhao et al., 1996). While several enzymes have been shown to catalyze these reactions (Vonesch et al., levels in vitro to maintain RA signaling activated as in the initial stage, leads to an immature lung phenotype characterized by failure to form typical distal buds. We show that this phenotype likely results from RA interfering with the establishment of a distal signaling center, altering levels and distribution of Fgf10 and Bmp4, genes that are essential for distal lung formation. Furthermore, RA upregulates P450RAI expression, suggesting the presence of feedback mechanisms controlling RA availability. Our study illustrates the importance of regional mechanisms that control RA availability and utilization for correct expression of pattern regulators and normal morphogenesis during lung development. 1994; Zhao et al., 1996; Romert et al., 1998), retinaldehyde dehydrogenases V1 and V2/RALDH-2 have a prominent role in generating RA. RALDH-2 expression is developmentally regulated in many organs (Niederreither et al., 1997; Moss et al., 1998; McCaffery et al., 1999). RALDH-2- /- mice in a RARE-lacZ transgene background (Rossant et al., 1991) do not produce sufficient RA to activate lacZ expression anywhere in the body, other than in the eye (Neiderreither et al., 1999). The early death and the major defects observed in these mutants not only confirm the importance of retinoid signaling in organogenesis, but they also suggest that RALDH-2 is a critical enzyme in the RA synthetic pathway. A balance between retinoid synthesis and degradation determines the RA concentration in target tissues. RA degradation in target cells may serve as a mechanism to protect RA-sensitive tissues from high RA levels. P450RAI (CYP26) is an RA-inducible RA-metabolizing enzyme of the cytochrome P450 family. P450RAI specifically converts RA into several hydroxylated products, limiting retinoid signaling in target cells or generating metabolites which differentially affect morphogenesis and regeneration (White et al., 1996; Fujii et al., 1997; Moss et al., 1998; Iulianella et al., 1999). Retinoid signaling can also be controlled by inhibition of receptor function. The Chicken Ovalbumin Upstream Promoter-Transcription Factors (COUP-TFs) are nuclear receptors that are believed to interfere with RA-mediated transactivation by sequestering RXRs. Because RXRs heterodimerize with members of the steroid receptor superfamily, including RARs, COUP-TFs act as inhibitors of RA actions (reviewed by Tsai and Tsai, 1997). COUP-TFs are expressed in a wide and partially overlapping domains during organogenesis. In the lung, only COUP-TFII has been reported (Jonk et al., 1994), and its role in lung development is still unclear. Targeted disruption of COUP-TFII gene leads to embryonic lethality by day 9-9.5 from defects in angiogenesis and heart development (Pereira et al., 1999). Here we perform an integrative study to understand how RA signaling is regulated during lung morphogenesis and how RA influences lung pattern formation. By mapping sites of retinoid synthesis (RALDH-2 expression), utilization (activation of a RARE-lacZ transgene) and degradation (P450RAI expression) and sites of expression of an RA signaling antagonist (COUP-TFII) in the embryonic lung, we identify distinct stages of RA signaling. We found that branching morphogenesis appears to require downregulation of RA signaling to allow proper expression of genes involved in distal lung formation, such as Fgf10 and Bmp4. Maintaining a status of RA activation characteristic of the early lung in organ cultures results in an immature lung phenotype characterized by failure to form typical distal buds. Our study illustrates the importance of regional mechanisms that regulate RA availability and utilization for correct expression of gene regulators of pattern and normal lung morphogenesis. MATERIALS AND METHODS Embryonic lung cultures Day-11.5 lungs isolated from CD-1 mouse embryos were cultured in BGJb control medium or in media containing all-trans retinoic acid (Sigma) at a final concentration of 10- 7 to 10- 5 M (Cardoso et al., 1995, 1996). Late exposure experiments consisted of culturing lungs in control medium for 3 days followed by another 3-day treatment with all-trans RA (10- 5 M). Lung cultures were harvested at days 1, 3 and 6 and fixed in 4% paraformaldehyde. In some experiments, ionexchange beads AG1-X2 (BioRad, Hercules, CA) soaked in RA solution (30 m M), or heparin beads soaked in FGF10 solution (human recombinant, 100 m g/ml, R&D) were grafted onto day-11.5 lung explants and cultured for 24-72 hours (Eichele (...truncated)