Supplemental Figure S1
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Fig. S1. Complementary data for MP1 and PMG identification, Nrx-IV in situ hybridization, and additional Nrx-IV rescue experiments. (A-D) Identification of MP1 neurons during embryogenesis. Developmental series showing the position of MP1 neurons (Lim3) with respect to Wrapper+ MG in sim-Gal4 UAS-tau-GFP embryos. A single MP1 neuron (arrow) is shown in each panel; the other MP1 neuron resides adjacent to the first, ∼4-5 µm away. (A) At stage 12, the MP1 neurons abutted the posterior side of a subset of AMG. (B) By stage 14, the AMG surrounded the AC, and the MP1 neurons were below the most ventral AMG. (C) At stage 15, while a dorsal-localized AMG moved over the PC (magenta asterisk), the MP1 neurons remained in contact with the most ventral AMG. (D) At stage 17, the MP1 neurons were still in contact with the ventral-most AMG. (E-H) Localization of PMG during embryogenesis. AMG were high wrapper RNA+, whereas PMG were En+ and low wrapper RNA+. (E) During stage 12, the PMG (arrows) migrated dorsally and toward the anterior. Only two PMG are shown in this focal plane. (F) By stage 13, one PMG (arrow) extended to the anterior to abut the posterior of the PC. (G) The PMG that touched the PC (arrow) remained in place during stages 15 and 16. (H) By stage 17, no sim-Gal4 UAS-tau-GFP+ Wrapper+ En+ cells were observed in the position of PMG; this is consistent with published reports that these cells undergo apoptosis (Sonnenfeld and Jacobs, 1995; Dong and Jacobs, 1997). (I-K) Nrx-IV RNA localization. Nrx-IV expression (purple) was assayed by whole-mount in situ hybridization. (I) Ventral view showing that Nrx-IV was expressed uniformly throughout the CNS and in peripheral glia along the nerves (arrows). (J) Sagittal view showing prominent expression in the: (1) PNS (arrow, also inset), (2) head region, including the foregut and salivary gland (bracket), and (3) hindgut (arrowhead). This pattern is identical to that previously published for Nrx-IV (Baumgartner et al., 1996). (K) Misexpression of UAS-Nrx-IV using sim-Gal4 showed high levels of expression in the midline cells. These experiments further confirm that the Nrx-IV in situ hybridization is specific and reinforce the notion that, unlike the protein, Nrx-IV RNA is not prominently expressed in midline cells. (L-O′) Nrx-IV rescue experiments. Sagittal views of two segments in embryos containing slit-Gal4 and UAS-Nrx-IV stained for Nrx-IV and Wrapper. (L-M′) In slit-Gal4 UAS-Nrx-IV embryos heterozygous for Nrx-IV (L,L′), an AMG was inserted between the AC and PC at stage 15 (white arrowhead) and Nrx-IV was localized in MG membranes (yellow arrowhead). (M,M′) At stage 17, AMG surrounded both the AC and PC. Nrx-IV staining in the epidermis (arrow) indicated that these embryos were not Nrx-IV mutant. (N-O′) In slit-Gal4 UAS-Nrx-IV embryos homozygous for Nrx-IV, Nrx-IV was observed at high-levels in MG at (N,N′) stage 15 and (O,O′) stage 17. These embryos show the Nrx-IV phenotype: (N,N′) at stage 15, an AMG nucleus was not interposed between the AC and PC and (O,O′) at stage 17, the PC was not ensheathed. Nrx-IV was not localized in MG membranes (yellow arrowhead in N,N′). The absence of anti-Nrx-IV staining in the epidermis (arrow) showed that these were Nrx-IV mutant embryos.
