DEV022343 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Representative images that document construction of the stage 10-11 molecular maps. Sagittal views of single segments of sim-Gal4 UAS-tau-GFP embryos; all images were from stage 11, except A,B,E,F, which were at stage 10. Column 1 (magenta), midline cell-type specific probe; (A-H) column 2 (blue), combined pdm2 and nubbin (nub) expression; (I-L) column 2, anti-Cas immunostaining; column 3, merge image showing all CNS midline cells (green) revealed by anti-GFP staining; column 4, schematic of expression. Pink corresponds to magenta gene expression, blue corresponds to blue gene expression, and purple indicates co-localization. Colored outlines represent cytoplasm staining, colored nuclei indicate nuclear staining, and absence of a nucleus indicates that the cell is undergoing division. (A) The undivided MP1,3,4 can be identified at late stage 10. The MP1 (white arrowhead), MP3 (yellow arrowhead) and MP4 (white arrow) are Embryonic lethal, abnormal vision (Elav)⁺ pdm2/nub⁺. (B) The MP4 (white arrow) can be distinguished from the MP3 (yellow arrowhead) and MP1 (white arrowhead) based on the presence of En. In this late stage 10 segment, MP4 can be seen dividing, based on dispersed localization of tau-GFP, while MP3 and MP1 are yet to divide. (C,D) Two populations of MG can be distinguished based on gene expression. (C) During stage 11, wrapper was present in the AMG and the MP1 (white arrowhead), but not other MPs and their progeny (bracket). MP1 wrapper expression diminished by late stage 11, while wrapper expanded to both AMG and PMG. (D) CG32244 was expressed in the PMG at stage 11. (E) The MP1 (white arrowhead) could be distinguished from the MP3 (yellow arrowhead) and the MP4 (white arrow) at late stage 10 based on Runt localization in MP1. (F) Anti-PH3 staining of a late stage 10 embryo showed a dividing MP4 (white arrow). The MP1 (white arrowhead), MP3 (yellow arrowhead) and MP5 (blue arrowhead) had not yet divided. (G) The MP1 neurons (white arrowheads) could be distinguished by expression of Runt. The VUM5 neurons were pdm2/nub^– (blue arrowheads), which distinguished them from the MP1, MP3 (yellow arrowheads) and VUM4 (arrows) neurons, which were pdm2/nub⁺. MP6 had not yet divided. (H) Segment showing the temporal sequence of MP cell division. The MP4 divided to give rise to two VUM4 neurons (white arrows) that were En⁺ pdm2/nub⁺. The MP3 (yellow arrowhead; En^– pdm2/nub⁺) was beginning to divide, whereas the MP1 (white arrowhead) and MP5 (blue arrowhead; En⁺ pdm2/nub^–) had not yet divided. (I) Segment in which the MP3 (yellow arrowheads) and MP4 (arrows) cells have divided, and MP5 (blue arrowhead) has initiated division. MP5 (Tkr⁺ Cas^hi) could be distinguished from the VUM4 neurons (Tkr^– Cas^lo) based on Tkr expression. Cas was transiently present at low levels in the MP6 (red arrowhead; Tkr⁺ Cas^lo) before the MP5 divided, and was absent after MP5 division. (J) MP5 (blue arrowheads; Tkr⁺ Cas^hi) division preceded MP1 (white arrowhead) and MP6 (red arrowhead; Tkr⁺ Cas^lo) divisions. The VUM4 neurons (white arrows) were Tkr^– Cas^lo and the MP3 neurons (yellow arrowhead) were Tkr^– Cas^–. (K) The MNB (red arrow; wor⁺ Cas^lo) was identified prior to its delamination and MP6 (red arrowhead; wor⁺ Cas^–) division. The MP3 (yellow arrowheads), VUM4 (white arrows) and VUM5 (blue arrowheads) neurons are shown, post-division. Only one of the two MP1 neurons (white arrowhead) is apparent in this focal plane. (L) All MPs have divided. Only one MP1 neuron (white arrowhead) is visible in this projection, and the MNB is also absent from this image. The MP3 (yellow arrowheads; Tkr^– Cas^–), VUM4 (white arrows; Tkr^– Cas^lo), VUM5 (blue arrowheads; Tkr⁺ Cas^hi), and VUM6 (red arrowheads; Tkr⁺ Cas^–) neurons are shown. Note that Cas was absent from the MP6 neurons.

• Supplemental Figure S2 -

  Fig. S2. Notch signaling is active in midline glia, MPs and midline neurons. (A,L) Sagittal views of single segments of stage 14 sim-Gal4 UAS-tau-GFP embryos are shown as confocal images. Midline cells are visualized by anti-GFP staining (green). (A,C,E,G) Wild type; (B,D,F,H) sim-Gal4 UAS-Su(H).VP16 misexpression embryos. (A) Immunostaining showed Runt (magenta) in wild-type AMG and MP1s (*). The AMG completely surround the anterior commissure (a) and partially surround the posterior commissure (p). (B) Misexpression of Su(H).VP16 resulted in excess anterior Runt⁺ cells (bracket). (C) Wild-type embryos showed high levels of Wrapper (magenta) in AMG (anterior cells) and lower levels in PMG (*). Brackets denote relative positions of AMG (white) and PMG (yellow). Inset corresponds to the yellow-bracketed area and shows Wrapper staining at low levels in PMG. (D) In sim-Gal4 UAS-Su(H).VP16, all midline cells were Wrapper⁺ with anterior cells Wrapper^hi, indicative of AMG (white bracket), and posterior cells Wrapper^lo (PMG, yellow bracket) indicative of PMG. Inset corresponds to yellow-bracketed area and shows Wrapper staining at low levels in all cells in this region. (E) In wild type, En (magenta) was present in PMG (arrowheads), as well as MNB, MNB progeny and iVUMs. (F) In sim-Gal4 UAS-Su(H).VP16, the number of En⁺ PMG was increased in the posterior region (yellow bracket). (G) In situ hybridization showed Cad74a expression (magenta) exclusively in wild-type PMG (arrowheads), and (H) the number of Cad74a⁺ cells was expanded in sim-Gal4 UAS-Su(H).VP16. (I) During stage 11, P12xSu(H)bs-lacZ was expressed at high levels in AMG, and a posterior cell cluster containing MP6, the MNB and PMG. MP5 (yellow arrowhead) expressed P12xSu(H)bs-lacZ at low levels. (J) After the MP5 division, low-level P12xSu(H)bs-lacZ expression was present in the VUM5 neurons (yellow arrowheads) and at a high level in MP6 (blue arrowhead). (K) After the MP6 division, P12xSu(H)bs-lacZ expression was present at low levels in the VUM5s (yellow arrowheads), medium levels in VUM6s (blue arrowheads) and high levels in the MNB (red arrowhead). (L) At stage 17, P12xSu(H)bs-lacZ was expressed in the MG (white arrowheads), the MNB and its progeny (pMNB, bracket), VUM5s (absent in this focal plane) and VUM6s (blue arrowheads). (M,N) Ventral views of stage 14 P12xSu(H)bs-lacZ (M) wild-type and (N) Dl embryos stained with anti-Sim (green) and anti-β-gal (magenta). The Dl mutant showed that midline and CNS expression of P12xSu(H)bs-lacZ was absent, indicating that P12xSu(H)bs-lacZ is dependent on N signaling. Expanded Sim⁺ muscle precursor cells (white arrowheads) and a few P12xSu(H)bs-lacZ⁺ cells (yellow arrowheads) with residual Dl protein (not shown) were present.

• Supplemental Figure S3 -

  Fig. S3. sim-Gal4/UAS-Su(H).VP16 females have reduced fertility and larval motility. (A) Ventral view of a sim-Gal4 UAS-tau-GFP third instar larval nerve cord showing midline neuronal axons visualized by anti-GFP staining. Horizontal bar indicates the midline. (B) In sim-Gal4 UAS-tau-GFP UAS-Su(H).VP16 larvae, there is a severe reduction in the number of midline neuronal axons, consistent with the absence of midline neurons observed in the embryo (see Fig. 3K-M). The effect is highly penetrant, and >90% of midline cells were absent. This allowed a worst-case test of the physiological and behavioral consequences of depleting all midline neurons. (C) Fertility was measured by scoring 20 crosses of each genotype for percentage vials with progeny. Individual flies were crossed to three w flies. The majority of the sim-Gal4 UAS-Su(H).VP16 embryos formed normal-appearing adults. Females showed low fertility, whereas males were relatively fertile. Previously, it was shown that octopaminergic/glutamatergic neurons residing along the CNS midline innervate the female genitalia and control oviduct contraction (Hardie et al., 2007; Monastirioti, 2003; Rodriguez-Valentin et al., 2006). These are likely to be mVUMs, and their loss would explain the female sterility. (D) The mean distance travelled (cm) in 5 minutes by larvae was measured for six genotypes. Wandering third instar larvae were removed from the walls of food containers and placed on a grape juice agar plate for 1 minute. Larvae were then transferred to a clean grape juice agar plate and allowed to move freely for 5 minutes. The indentations in the grape juice agar plate caused by moving larvae were scanned on a flatbed scanner and analyzed for distance travelled using ImageJ software. The larval path was traced using the Freehand Lines tool and the distance travelled expressed in cm. For each genotype, distances travelled for 16-25 individual larvae were averaged and the s.d. (error bars) calculated. The number and identity of midline neurons and glia were wild-type in each of the genotypes examined, with the exception of sim-Gal4/UAS-Su(H).VP16. The mean distance travelled by sim-Gal4/UAS-Su(H).VP16 larvae was reduced compared to the controls: sim-Gal4/+ (2-fold reduction) and UAS-Su(H).VP16/+ (3-fold reduction). The larval locomotory defects resemble abnormalities associated with octopaminergic mVUM control of larval body wall muscles and movement (Nishikawa and Kidokoro, 1999; Saraswati et al., 2004). Thus, the results with sim-Gal4 UAS-Su(H).VP16 animals were validated by other studies, and suggest that the remaining neurons, although likely to be important, do not have dramatic effects on behavior or physiology. Use of a sim temperature-sensitive strain previously showed that reduction in sim function in the late embryo resulted in adult female sterility and defects in locomotion (Pielage et al., 2002). However, in these flies, it is unknown whether midline neurons, including mVUMs, are present and functional and thus contribute to these phenotypes. By contrast, defects in sim^ts male and female genitalia, gonadogenesis and male courtship behavior could lead to sterility, and the locomotory defects could be due to defects in the central brain.

  References

  Hardie, S. L., Zhang, J. X. and Hirsh, J. (2007). Trace amines differentially regulate adult locomotor activity, cocaine sensitivity, and female fertility in Drosophila melanogaster. Dev. Neurobiol. 67, 1396-1405.

  Monastirioti, M. (2003). Distinct octopamine cell population residing in the CNS abdominal ganglion controls ovulation in Drosophila melanogaster. Dev. Biol. 264, 38-49.

  Nishikawa, K. and Kidokoro, Y. (1999). Octopamine inhibits synaptic transmission at the larval neuromuscular junction in Drosophila melanogaster. Brain Res. 837, 67-74.

  Pielage, J., Steffes, G., Lau, D. C., Parente, B. A., Crews, S. T., Strauss, R. and Klambt, C. (2002). Novel behavioral and developmental defects associated with Drosophila single-minded. Dev. Biol. 249, 283-299.

  Rodriguez-Valentin, R., Lopez-Gonzalez, I., Jorquera, R., Labarca, P., Zurita, M. and Reynaud, E. (2006). Oviduct contraction in Drosophila is modulated by a neural network that is both, octopaminergic and glutamatergic. J. Cell. Physiol. 209, 183-198.

  Saraswati, S., Fox, L. E., Soll, D. R. and Wu, C. F. (2004). Tyramine and octopamine have opposite effects on the locomotion of Drosophila larvae. J. Neurobiol. 58, 425-441.

• Supplemental Figure S4 -

  Fig. S4. numb and spdo regulate axonal trajectory. Composite confocal images of single segments from stage 15 (A,E) wild-type, (B,F) numb⁴/numb⁴, (C,G) spdo^G104/spdo^G104 and (D,H) sim-Gal4 UAS-numb embryos in ventral view with anterior to the left. All embryos had sim-Gal4 UAS-tau-GFP (green) in the background to visualize all midline cells and their axons. The midline axons can be discerned based on their characteristic positions along the anterior-posterior and dorsal-ventral axes. For clarity, non-relevant axons have been subtracted and relevant axons have been pseudocolored. MP1 axons were not significantly affected in any of the mutant embryos. (A-D) MP3 neurons. H-cell sib (yellow), H-cell (magenta), MP1 neurons (green). (A) In wild type, the H-cell sib axon bifurcated in the anterior commissure and sent projections on both sides of the midline in an anterior direction. The H-cell axon bifurcated in the posterior commissure and sent projections into the longitudinal tract lateral to the MP1 axons. The MP1 axons emanated from the lateral face of the cell body and extended posteriorly to the longitudinal tract where they bifurcated and sent axons in both anterior and posterior directions. (B) In numb, the H-cell sib axons were present and appeared thickened while the H-cell axons were absent. (C) In spdo, the H-cell sib axon was absent while the H-cell axons were duplicated and characteristically extended past the MP1 axons. (D) Overexpression of numb in all midline cells resulted in the absence of H-cell sib axons, but possessed thickened H-cell axons similar to the spdo mutant phenotype. (E-H) VUM neurons. mVUMs (yellow), iVUMs (magenta), MP1 neurons (green). (E) In wild type, the mVUM axons bifurcated in the anterior commissure and projected along the segmental and intersegmental nerves into the muscle fields. The iVUM axons bifurcated in the posterior commissure and extended projections anteriorly within the longitudinal tracts. (F) In numb, the iVUM axons were present while the mVUM axons were absent. (G) In spdo, the iVUM axons were absent while the mVUM axons were present and appeared thicker. (H) numb overexpression resulted in the presence of thickened mVUM axons and the absence of iVUM axons.

• Supplemental Figure S5 -

  Fig. S5. numb and spdo are not required for MP1 neuronal fate. Confocal images of single midline segments in stage 14 sim-Gal4 UAS-tau-GFP embryos. Anterior is to the left and all views are ventral. (A,E) Wild type, (B,F) numb⁴/numb⁴, (C,G) spdo^G104/spdo^G104, (D,H) sim-Gal4 UAS-numb. (A-D) Anti-Lim3 immunostaining (magenta) shows prominent localization to the MP1 neurons (arrowheads) in (A) wild-type, (B) numb, (C) spdo and (D) sim-Gal4 UAS-numb embryos. (E-H) Pigment-dispersing factor (Pdf) (magenta) is expressed in both MP1 neurons (arrowheads) in (E) wild-type, (F) numb, (G) spdo and (H) sim-Gal4 UAS-numb embryos. MP1 axonal trajectories (arrows) were not altered in mutant or overexpression backgrounds.

• Movie 1 -

  Movie 1. Time-lapse imaging of MP and MNB divisions. Live sim-Gal4 UAS-tau-GFP embryo imaged for GFP fluorescence for 144 minutes during stages 10-11. Sagittal view of one segment is shown (brackets), with anterior (Ant) to the left and interior (Int) at top. The movie pauses periodically to indicate the appearance of MPs (arrowheads) and their neuronal progeny (arrows), followed by the stem cell division of the MNB (arrowhead) into an MNB and GMC (arrow).

• Movie 2 -

  Movie 2. Time-lapse imaging of Dl mutant embryos. Sagittal view of an 80-minute excerpt from a stage 11 sim-Gal4 UAS-tau-GFP; Dl³/Dl³ embryo that was imaged for GFP fluorescence for 127 minutes total. Anterior (Ant) is to the left and interior (Int) at top. The midline cells are pushed inward by the hypertrophied lateral CNS. Note near-simultaneous divisions occurring in neighboring cells.