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Fig. 5. numb and spdo control MP3 neuronal cell fate. Confocal images of stage 14-15 Drosophila embryos in sagittal view. (A-E,P) Wild type, (F-J) numb⁴/numb⁴, (K-O) spdo^G104/spdo^G104, (Q) sim-Gal4 UAS-numb and (R) sim-Gal4 UAS-spdo. All embryos had sim-Gal4 UAS-tau-GFP (green) in the background, except H, which shows anti-Sim (green) staining. To identify the MP3 neurons, numb mutants were double-labeled with Vesicular glutamate transporter (VGlut) (not shown, except in I); spdo mutants were double-labeled with ple (not shown, except in L). In A-O, white arrowheads denote cells expressing H-cell genes and yellow arrowheads indicate cells expressing H-cell sib genes. (A,B) In wild type, Tailup (Tup) protein and ple were present in the H-cell, and absent from H-cell sib. (F,G) In numb, Tup and ple were absent from both MP3 neurons. (K,L) In spdo, Tup and ple were present in both MP3 neurons. (C) In wild type, fork head (fkh) was expressed in H-cell sib, the two MP1 neurons (^*) and iVUMs; only one iVUM (arrow) is present in this focal plane. (H) In numb, fkh was expressed in two Sim⁺ MP3 neurons, and was absent (M) from spdo MP3 neurons. (D) In wild type, VGlut was expressed in H-cell sib and at a lower level in mVUMs (black arrowheads), whereas (I) in numb, VGlut was expressed in two MP3 neurons and absent from VUM neurons. By contrast, (N) in spdo, the two MP3 neurons (arrowheads) lacked VGlut, whereas it was present in all VUM neurons. (E) pdm2 was expressed in the MP1 (^*) neurons and in both MP3 neurons in wild type (only one MP1 neuron is present in this focal plane). The expression of pdm2 was unaltered in (J) numb and (O) spdo. (P-R) Overexpression of numb, but not spdo, causes an MP3 cell fate change. The H-cell is marked by ple expression (magenta) and H-cell sib by CG13565 expression (blue). (P) Wild-type expression of ple and CG13565. (Q) In sim-Gal4 UAS-numb, H-cell sib was transformed into an H-cell, as shown by the presence of two ple⁺ cells and the absence of CG13565-expressing cells. (R) sim-Gal4 UAS-spdo showed a wild-type pattern of gene expression with a single ple⁺ cell and a single CG13565⁺ cell.

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