Fig. 4. Characterizing jag nub patterning defects in the anthers using FIL, PHB and SPL expression. (A-D) Expression of FIL detected by in situ hybridization using an anti-sense probe. (A) During wild-type stamen development, FIL is initially expressed throughout the abaxial side at stage 6. (B) As the adaxial microsporangia proliferate, FIL-expressing tissues develop into the connective and grow more slowly in comparison to the adaxial region. Similar to wild type (C), FIL expression in stage 6 jag nub stamens (D) is observed throughout the abaxial half. Although the FIL expression domain is soon dwarfed by the proliferation of the microsporangia in wild type, in jag nub anthers, no microsporangia proliferation is apparent and the FIL expression domain extends towards the adaxial side. (E,F) Expression of PHB detected by in situ hybridization using an anti-sense probe. (E) In wild type, PHB expression is detected in cells marking the dehiscence zone in between the two microsporangia in each pair (black arrowhead), as well as in the vasculature (red arrowhead). (F) In jag nub mutants, PHB expression is only detected in the vasculature. (G-K) SPL expression monitored using the SPL::GUS reporter. In wild type, SPL-reporter activity marks two domains in the anther that develop into the two sets of microsporangia (G, stage 6 shown, white arrowheads) and is maintained in the anthers until anthesis (H, stage 12 shown). (I) In jag nub mutants, the SPL reporter is activated in a similar spatial pattern to wild type. SPL-reporter activity is maintained in jag nub anthers until about stage 11 (J), but then disappears by anthesis (K, stage 12 shown). ab, abaxial side; ad, adaxial side; co, connective; gy, gynoecium; ms, microsporangia. Scale bars: 50 µm.