neuralized regulates Sanpodo protein localization. Sanpodo protein (red) and Delta protein (blue) localization in neur^1F65 mutant clones (green). (A–C) A region of the pupal thorax containing both wild-type and neur^1F65 mutant tissue, to the left, a wild-type pIIa/pIIb cell pair shows strong accumulation of Sanpodo (red) and Delta (blue) in cytoplasmic puncta that can colocalize (arrowhead), in the pIIb cell. At the right, the GFP+ neur^1F65 mutant tissue (green) exhibits a large cluster of cells that have high levels of Sanpodo (red) primarily at the membrane, as well as in cytoplasmic puncta, and accumulation of Delta (blue) at the cell surface.

lgl specifically regulates the membrane localization Sanpodo, but not the endocytosis of Delta. shibire regulates the cytoplasmic localization of Sanpodo. (A–C) A two-cell cluster of ES lineage cells in a shi^ts1 mutant pupae 22 h APF maintained at 22°C (A) and 32°C (B) or 37°C (C) labeled with anti-Sanpodo antibody (red) and pI, pIIb, and pIIa cell marker Asense (blue). At 22°C, Sanpodo protein is found in cytoplasmic puncta in the pIIb cell (white arrowhead). Inactivation of shibire (B and C) causes an accumulation of Sanpodo at the membrane. At 32°C (B), small cytoplasmic puncta and membrane-associated Sanpodo are observed (arrowhead), whereas at 37°C (C), Sanpodo is primarily at the membrane in both pI and pIIa/pIIb cell pairs as labeled by Asense (lower magnification view of one pI cell and three pIIa/pIIb cell pairs). (D–E) lgl is dispensable for the endocytosis of Delta into the pIIb cell. Endocytosis of Delta can be assayed by culturing nota in serum containing antibodies raised against the extracellular domain of Delta (Le Borgne and Schweisguth, 2003). Outside the mutant clone, control pIIa and pIIb cell clusters (D) internalize extracellular Delta (Dl^endo, red) into the pIIb cell (white arrowhead). Similarly, Endocytosed Delta (arrowheads, red) is detected as large puncta that are labeled with anti-Delta antibodies (arrows, blue) in the pIIb cell in lgl⁴ (E) MARCM mutant ES clusters (green).

Sanpodo is required for Notch-mediated fates in the adult PNS. (A) A wild-type adult thorax. (B) Adult thorax containing a large sanpodo^C55 Sb mutant clone; patches of the notum are bald, lacking external sensory structures. (C) Z-projection of MARCM clones of sanpodo^C55 Sb, in a 24-h (C) and a 28-h (D) APF (after pupae formation) pupa. (C) Outside the clone (approximate clone border marked by dashed line), external sensory organs contain one neuron (arrowhead), which expresses ELAV (red), whereas in sanpodo mutant clusters (marked by GFP, green) more than two cells expresses ELAV. (D) Sanpodo mutant cells (green) form clusters of ELAV-positive (red) neurons. (E) A mitotic pI cell labeled with Sanpodo (red) and Numb (green) antibodies. Images taken from pupae collected at 22 h APF. Sanpodo is uniformly distributed along the cell membrane and is also present in large puncta in the cytoplasm. Sanpodo does not strongly colocalize with Numb at the anterior cortex. (F) A schematic representation of a pIIa and pIIb cell cluster the gray dashed line indicates the depth of the XY plane shown in H, with Sanpodo represented in red. An XZ plane (G) and an XY plane (H) through the same wild-type pIIa/pIIb cell cluster. Sanpodo is present in strongly labeled apical cytoplasmic puncta (white arrowheads) in the pIIb cell and is detectable at the cell membrane (white arrow) in the pIIa cell.

Regulation of Sanpodo localization by lgl, numb, and α-adaptin. (A–C) Sanpodo protein labeled with anti-Sanpodo antibody (red) and mutant clone tissue is labeled with mCD8-GFP (green) in MARCM clones. A two-cell cluster of lgl⁴ (A), nb² (B), α-adaptin^ear5 (ada^ear5; C), in mutant cells (green); Sanpodo protein (red) is strongly enriched at the membrane in both cells and cytoplasmic puncta of Sanpodo are reduced or absent (see Figure 1, G and H, for Sanpodo protein in wild-type pIIa and pIIb cells). (A–G) Images taken from pupae collected at 22 h APF. (D and E) Lgl protein labeled with anti-Lgl antibody (red), and clone tissue is labeled with mCD8-GFP (green) in MARCM clones, and pIIa and pIIb cells are labeled with Asense (blue). Outside the mutant clone (D), Lgl protein localizes to the cortical region of both pIIa and pIIb cell (white arrow). (E) A two-cell cluster (green) within a clone overexpressing both mCD8-GFP and aPKC^ΔN, Lgl protein (red) is distributed throughout in the cytoplasm, but is largely absent from the cortical region (white arrow). (F and G) Sanpodo protein labeled with anti-Sanpodo antibody (red), and clone tissue is labeled with mCD8-GFP (green) and overexpressed PKC^ΔN (blue) in MARCM clones. (F) A pIIa and pIIb cell cluster (green) in clone tissue overexpressing GFP, Sanpodo (red) is present in strongly labeled cytoplasmic puncta (arrowheads) in the pIIb cell and is present at the cell membrane and in the cytoplasm in the pIIa cell, and low levels of endogenous PKC^ΔN (blue) are detected. (G) A two cell cluster (green) within a clone overexpressing both mCD8-GFP and PKC^ΔN, Sanpodo protein is strongly enriched at the membrane in both cells, specifically at the cell-cell border (arrowhead); little Sanpodo protein is present in the cytoplasm, and the overexpressed PKC^ΔN (blue) is detected in both cells. (H and I) Adult external sense organs overexpressing mCD8-GFP (Neur-Gal4>U-mCD8-GFP) or overexpressing both mCD8-GFP and activated PKC^ΔN (Neur-Gal4>U-mCD8-GFP>U-PKC^ΔN) in MARCM clones. Adult ES organs overexpressing the transgenes are marked by the presence of dark (y+) bristles and are surrounded by epidermal hairs exhibiting the ck phenotype (multiple epidermal hairs). (H) Overexpression of mCD8-GFP alone results in ES organs that develop into single bristle cell, single socket cell; which is characteristic of wild-type ES organs (see light colored y–bristles outside the ck marked clone). (I) Overexpression of both mCD8-GFP and PKC^ΔN results in organs developing supernumerary socket cells (black arrowheads) within the y+ ck marked clone.