Figure 7. Bmper inhibits Bmp signaling during lung development. Lung development and Bmp4 activity were evaluated in wild-type (WT) mice versus mice with a heterozygous (HT) inactivation of Bmper at postnatal day 0. (a) Hematoxylin and eosin analysis of heterozygous lungs demonstrated delayed branch- ing morphogenesis and inadequate expanded alveoli, which were separated by expansive interstitial mesen- chyme. (b) Staining for the type II epithelial cell marker prosurfactant C (arrows). Bars, 100 μM. Positive stain- ing is brown and counterstaining is blue. Graphical representation of the percentage of prosurfactant C– positive cells in wild-type versus Bmper het lungs. (c) Id1 mRNA expression was measured as a target of downstream Bmp activity by PCR. (d) Representative blots of endogenous Bmp4 in lysates (postnatal day 0) of lung for two individual animals each of wild-type, heterozygous, and knockout (KO) Bmper mice. freshly isolated day 15.5 lung mesenchyme with Bmp4 (1 nM) along with molar concentrations of Bmper that enhanced (0.5 nM) or inhibited (50 nM) Bmp4-mediated Smad activation in MECs (Fig. 6 c). As anticipated, Bmp4 treatment alone pro- tected lung MEFs from a default program of apoptosis as mea- sured by cleaved caspase-3 (Fig. 6, c and d). At concentrations below those of Bmp4, Bmper enhanced these antiapoptotic ef- fects of Bmp4, whereas when the molar concentration of Bmper exceeded Bmp4, Bmper attenuated Bmp4 activity, restoring apoptosis in these cells. Consistent with our concentration- dependent hypothesis, lower concentrations of Bmper alone were more protective than higher concentrations. The fact that we were able to demonstrate a dynamic func- tional relationship between BMPER and Bmp4 in lung MEFS suggested the very real possibility that this novel, concentration- dependent relationship between Bmper and Bmp4 may have physiological importance, especially in the case of organ devel- opment that requires Bmp4 activity, such as the lungs (Weaver et al., 1999; Weaver et al., 2000). To test this possibility and determine the physiological importance of Bmper, we engi- neered Bmper-deficient mice by homologous recombination (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb .200808064/DC1). Bmper mice died at birth (similar to the findings of Ikeya et al., 2006), whereas heterozygotes survived. Both Bmper +/ and Bmper / mice exhibited widespread tissue- specific defects during embryonic development. Given the important role of Bmp4 signaling in lung development and our findings that Bmp4 and Bmper share a concentration-dependent relationship in the regulation of lung MEF survival we were particularly interested in examining the structural defects in the lungs of Bmper-deficient mice. In the perinatal lung of Bmper +/ mice, an overgrowth of mesenchymal cells (which nor- mally express Bmper) appeared to prevent complete alveoli ex- pansion, as indicated by smaller and fewer terminal sacs that were separated by thickened interstitial mesenchyme (Fig. 7 a). These anatomical defects are similar to lung phenotypes re- ported previously in Bmper-deficient mice (Ikeya et al., 2006). Upon closer examination of the cellular and molecular features resulting from a decrease in Bmper expression, we de- tected several anomalies that were consistent with a “pro-Bmp” effect in the Bmper +/ lungs. At embryonic day 18.5, there was an increased ratio of type II epithelial cells to total distal cells (Fig. 7 b) as well as fewer apoptotic cells detected by TUNEL assay in Bmper +/ lungs compared with wild-type mice (not depicted). This abundance of interstitial mesenchymal tissue was not caused by altered differentiation or increased cell proliferation as neither of these parameters were significantly altered in Bmper +/ lungs compared with wild-type litter- mates. Not surprisingly, this observed phenotype was heavily localized to the distal regions of the developing lung, the region in which lung mesenchyme usually regresses if Bmp signaling 604 JCB • VOLUME 184 • NUMBER 4 • 2009