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First published online 13 August 2008
doi: 10.1242/dev.022343

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Multiple Notch signaling events control Drosophila CNS midline neurogenesis, gliogenesis and neuronal identity

Department of Biochemistry and Biophysics and Department of Biology, Program in Molecular Biology and Biotechnology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Formation of midline precursors (MPs) and MP neurons in Drosophila. Molecular map of stage 10 and 11 MPs and midline neurons (circles) and glia (ovals) shown in sagittal view. One segment is shown, with anterior to the left and interior (basal) at top. Each cell is depicted in terms of its pattern of gene expression as indicated by colors (the corresponding genes as listed on the left). The five MPs are shown at late stage 10, and the arrows indicate MPs dividing into their neuronal progeny at stage 11. The number of midline glia does not change appreciably from stage 10 to 11.

View larger version (45K): [in this window] [in a new window]   Fig. 2. Time-lapse imaging of sequential MP delamination and division. Images in sagittal view, internal (basal) up, from time-lapse imaging of an (A-E) MP6 division and an (F-J) MP1 division. GFP fluorescence was visualized in sim-Gal4 UAS tau-GFP embryos during stage 11. Time is displayed as minutes:seconds. Relevant cells in each panel are pseudocolored. (A) Prior to division, the MP6 (white arrowhead) delaminates from the apical surface and takes on a triangular shape. The tip of the retracting cell is indicated by the yellow arrowhead. (B-D) During mitosis, (B) the centrosomes (arrows) move toward opposite poles, (C) the spindle fibers have an apical-basal orientation, and (D) the MP6 divides (arrowheads) along this axis. (E) Two MP6 neurons (arrowheads) are produced. (F) The MP1 (white arrowhead) delaminates from the apical surface, also acquiring a triangular shape (retraction point, yellow arrowhead). (G) The centrosomes (arrow) can be seen just before they separate and begin their migration. (H) The MP1 spindle maintains an orientation perpendicular to the apical-basal axis. (I,J) Cytokinesis results in the formation of two MP1 neurons (arrowheads).

View larger version (102K): [in this window] [in a new window]   Fig. 3. Notch signaling influences midline cell fate. Ventral views of (A-E) wild-type, (F,G,I,J) Dl3/Dl3, (H) Dl3/Dl7 and (K-O) sim-Gal4 UAS-Su(H).VP16 stage 14 Drosophila embryos. Cell types are listed at the top of each column, and the gene or protein assayed that identifies each cell type is listed below. Horizontal bars indicate the location of the midline. (F,I) To differentiate (F) Odd+ and (I) wor+ midline cells from lateral CNS cells, embryos were double-stained with anti-Sim [not shown, but outlined (dashed line) to show location of midline cells]. In Dl mutants, there was an (F-H) increase in MP1, MP3 (H-cell) and mVUM neurons, and an absence of the (I) MNB and (J) MG. (G) Ectopic ple+ cells (arrowheads) were present off the midline; double-staining with anti-Sim indicated that these are not midline-derived (not shown). (K-O) sim-Gal4 UAS-Su(H).VP16 embryos showed the opposite phenotype to Dl mutants: (K-M) strong reduction of MP1, MP3 and mVUM neurons, and increases in (N) MNB and (O) MG.

View larger version (125K): [in this window] [in a new window]   Fig. 4. MP number increases in the absence of Notch signaling. (A-D') Ventral views of (A-B') wild-type and (C-D') Dl3/Dl3 mutant stage 14 Drosophila embryos. (A,A',C,C') Single segments stained for Tkr (magenta) and Tbh (green). (A,A') The mVUM6 (blue arrowhead) was Tkr+, whereas mVUM4 (black arrowhead) and mVUM5 (yellow arrowhead) were Tkr-. Dotted ovals outline the mVUMs. (C,C') In Dl, Tkr expression was absent indicating that the excess Tbh+ cells were not mVUM6s. (B,B',D,D') Single segments stained for Cas (magenta) and Tbh (green). (B,B') There are three Tbh+ mVUMs in each segment: mVUM4 (black arrowhead) was Caslo, mVUM5 (yellow arrowhead) was Cashi, and mVUM6 was Cas- (blue arrowhead). (D,D') Excess Tbh+ cells in Dl mutants were Caslo, indicating that they were mVUM4s. (E-H') Sagittal views of single segments of (E) wild-type and (F-H') Dl3/Dl3 mutant embryos. Midline cells are defined as MPs based on their presence at stage 10 and relatively large size. (E) At mid-stage 10, there is a single Odd+ (magenta) MP1 (arrowhead). (F) In Dl, the number of Odd+ MP1s (bracket) was increased. (G,G') Dl mutant embryo at two focal planes, 8 µm apart, showing three dividing cells (arrowheads 1-3) in close proximity, stained with anti-phosphohistone H3 (PH3, magenta). (H,H') In Dl, there is an increase in Odd+ (magenta) Cas+ (blue) MP1 neurons (magenta bracket), Odd- Cas- MP3 neurons (white bracket), and Odd- Cas+ MP4 neurons (blue bracket).

View larger version (79K): [in this window] [in a new window]   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) spdoG104/spdoG104, (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.

View larger version (75K): [in this window] [in a new window]   Fig. 6. numb and spdo control VUM 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) spdoG104/spdoG104, (Q) sim-Gal4 UAS-numb and (R) sim-Gal4 UAS-spdo. All embryos had sim-Gal4 UAS-tau-GFP (green) in the background, except I, which shows anti-Sim (green) staining. To identify the VUM neurons, numb mutants were double-labeled for En (not shown, except in H); spdo mutants were double-labeled with Tbh (not shown, except in L). In A-O, white arrowheads indicate cells expressing mVUM genes and yellow arrowheads indicate cells expressing iVUM genes. (A) In wild type, Zn finger homeodomain 1 (zfh1) was present in all three mVUMs and not in the iVUMs. (F) In numb, zfh1 expression was absent. (K) In spdo, zfh1 expression was expanded to five VUMs. (B) Tbh was expressed in three mVUMs in wild type. (G) In numb, Tbh was not expressed. (L) In spdo, five VUMs expressed Tbh. (C) In wild type, En was present in three iVUMs as well as other cell types, including the PMG (bracket). (H) In numb, En was present in five VUMs in addition to the PMG (bracket). (M) In spdo, En was absent from VUMs, but was present in the PMG (asterisk and bracket). (D) Glutamic acid decarboxylase 1 (Gad1) was expressed in three iVUMs in wild type. (I) Gad1 expression was expanded to six cells in numb (four of the six VUMs can be seen in this focal plane). (N) In spdo, Gad1 expression was present in only one VUM. (E) In wild type, Cas was present in two iVUMs (iVUM4,5) and two mVUMs (mVUM4,5). In (J) numb and (O) spdo mutant embryos, Cas was also present in iVUM4,5 and mVUM4,5. (P-R) Overexpression of numb causes a VUM cell fate change. (P) Wild-type expression of Tbh (magenta) in three mVUMs and of En protein (blue) in three iVUMs. (Q) In sim-Gal4 UAS-numb, six ventral Tbh+ En- mVUMs (two of the six cells are absent in this focal plane) were present. En in PMG (bracket) was unaffected. (R) sim-Gal4 UAS-spdo had a wild-type Tbh and En pattern (two of three Tbh+ mVUMs are present in this image).

View larger version (66K): [in this window] [in a new window]   Fig. 7. Numb and Spdo localization during MP divisions. Confocal images of MP1 and MP4 divisions in sim-Gal4 UAS-tau-GFP (green) stage 11 Drosophila embryos stained with (A-F) anti-Numb (magenta) and (G-L) anti-Spdo (magenta). Sagittal views with anterior left and internal (basal) up. White arrowheads indicate: (A,B,G,H) MP4, (C,D) Numb+ VUM4 neuron, (I,J) basal VUM4 neuron with cytoplasmic punctate Spdo, (E,K) MP1 and (F,L) MP1 neurons. Yellow arrowheads indicate: (C,D) Numb- VUM4 neuron, (I) apical VUM4 neuron with cytoplasmic punctate Spdo and (J) membranous Spdo VUM4 neuron.

View larger version (42K): [in this window] [in a new window]   Fig. 8. Model of Notch regulation of midline cell fate in Drosophila. (A) Mesectodermal cells meet at the ventral midline before the stage 8 1414 division. Little is known regarding influences on midline cell development at this stage. (B) After the stage 8 division, but before Notch signaling, the 16 midline cells can be considered as three equivalent groups of cells: MP1s, MP3s and MP4s. (C) After Notch signaling, the 16 cells acquire specific fates, and differ in their levels of Notch signaling as indicated by the expression of Su(H)-lacZ reporter. For simplicity, midline cells are shown as paired cells along the anterior-posterior axis. The precise anterior-posterior and left-right positions of individual cells are unknown, except that AMG, MP1 and MP3 tend to reside in the anterior half, and PMG, MP5, MP6 and MNB in the posterior half. Different shades of blue indicate relative levels of Notch signaling. (D) Asymmetric cell division. Notch signaling is required for MP3-6 asymmetric cell fates. Notch signaling is active (blue diagonals) in H-cell sib and iVUMs, and inhibited in H-cell and mVUMs. Assayed genes expressed in the MP3 and VUM lineages are shown below each neuron: bold text indicates expression, whereas the light, shaded text indicates repression. Genes are categorized as either Notch-activated, Notch-repressed or Notch-independent. Functional classes of genes are color-coded: transcription factors (blue), neural function genes (red) and others (black).

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