Branching restarts on removal of IQ1. (A) E13.5 lung explants were cultured with IQ1 for 48 hours. Subsequently, the IQ1 was removed from the culture media. After two days of the additional culture without IQ1, new branching ends were found at the distal tips (arrows). (B) E11.5 embryos were treated with IQ1 in utero for one day. The embryos were subsequently dissected at E14.5 after two days of growth in the absence of IQ1. ALP staining of the left lobe shows fine branching equivalent to the control although the lobe is smaller. (C) E13.5 lung explants were cultured with either DMSO (control) or IQ1 (10 μM) for 24 hours. Immunohistochemistry of α-SMA shows myofibroblasts surrounding the proximal airway. However, myofibroblasts are absent at the distal tips in both the control and IQ1 treated lung explants (arrows). Nuclei were stained with hematoxylin.