FIGURE 2.

Inhibiting γ-secretase activity in the foregut disrupts proximal-distal pattern of the lung primordium. A and B, WMISH of Titf1 in control foregut cultures (E8.5 + 3 days) shows strong signals in lungs buds (Lu) distally (arrowhead) and in the thyroid primordium (Th). DAPT treatment (50 μm) of foreguts results in expansion of the Titf1-expressing lung region (double arrowheads) and shortening of the proximal region. C and D, in controls, Sox2 labels the foregut endoderm, including the proximal lung (bracket), which is markedly truncated in DAPT-treated explants (small bracket). E, Western blot of cleaved Notch (NICD) shows loss of expression in DAPT-treated foreguts. F-H, WMISH of Hey2 in control foregut culture shows strong signals in heart (Ht) and in the foregut endoderm, including the primary lung buds. G and H, in DAPT-treated foreguts, Hey2 expression is selectively abolished in heart and lung buds but not elsewhere in the foregut. The severity of the lung phenotype correlates with the overall degree of Hey2 down-regulation in DAPT-responsive tissues (compare signals in the heart; green arrows). I and J, Hes5 is present in the lung and stomach regions of the control foregut, and expression is abolished by DAPT. However, Hes1 expression in distal lung buds seems unchanged after DAPT treatment (K) (data not shown). n = 3-6 foreguts/condition in both WMISH and Western blot experiments. Bar (A), 300 μm.