Immunostaining

Tissue was blocked for an hour at room temperature or overnight at 4 °C in phosphate buffered saline with 2% Normal Donkey Serum (Jackson ImmunoResearch), followed by 2 hr at room temperature in primary antibodies, and 1 hr at room temperature in secondary antibodies. Primary antibodies include: rabbit anti-Eve (1:1000, see Meng et al., 2019), chicken anti-GFP (1:1000, Aves #GFP-1020), rat anti-Worniu (1:250 Abcam #ab196362), guinea pig anti-Runt (1:500, John Rientz, UChicago), guinea pig anti-Hb (1:1000, John Rientz, UChicago), guinea pig anti-Kruppel (1:1000, John Rientz, UChicago), rat anti-Zfh2 (1:800 Chris Doe, UOregon), rabbit anti-Castor (1:1000 Chris Doe, UOregon), guinea pig anti-HB9 (1:1000 Heather Broiher, Case Western), rabbit anti-Smad3 (pMad) (1:300 Abcam #52903). The following monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA: mouse anti-Brp (1:50, NC82), mouse anti-DLG (1:500, 4F3), mouse anti-GluRIIA (1:25, 8B4D2), mouse anti-En (1:5, 4D9), mouse anti-Eve (1:50, 3C10), mouse anti-Cut (1:50, 2B10). Secondary antibodies were from Jackson ImmunoResearch and were reconstituted according to manufacturer’s instructions and used at 1:400. 647-Phalloidin (1:600, Thermofisher A22287) Rhodamine-Phalloidin (1:600, Thermofisher R415), Cy3-HRP (Jackson ImmunoResearch 123-025-021), 647-HRP (Jackson ImmunoResearch 123-605-021). Embryos were staged for imaging based on morphological criteria. Whole mount embryos and larval fillets were mounted in 90% Glycerol with 4% n-propyl gallate. Larvae brain preparations were mounted in DPX (Sigma-Aldrich, St. Louis, MO).