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. 2019 Mar 26;8:e41801. doi: 10.7554/eLife.41801

Figure 5. Qsox1 is required for macrophage dissemination and entry into the germband tissue.

(A) Representative confocal images of early Stage 12 embryos from control and P{SUPor-P}Qsox1KG04615 = qsox1KG04615. (B–C) Quantification in early Stage 12 embryos showing a significant reduction in germband macrophages (B) in the P-element mutant qsox1KG04615 located in the Qsox1 5’UTR (n = 18, p=0.0012) and (C) upon the expression in macrophages under srpHemo-GAL4 control of an RNAi line (v108288) against Qsox1 (n = 24, 23 embryos, p=0.001). (D) Images from two-photon movies from control and qsox1KG04615. Macrophage nuclei (red) are labeled with srpHemo-H2A::3xmCherry. Stills at 0, 60, 90, 120, 150 and 180 min. (E) Quantification of macrophage speed reveals 18% slower macrophage migration in the head towards the yolk neighboring the germband in the qsox1KG04615 mutant compared to the control (n = 3 movies for each, #tracks: control = 329, mutant = 396, p=0.0056). (F) Quantification of the time required for macrophage entry into the germband in qsox1KG04615 compared to the control. n = 3 movies for each, p=0.043. (G) Quantification of macrophage speed in the germband in the qsox1KG04615 mutant compared to the control (n = 3 movies for each, #tracks: control = 21, mutant = 19, p=0.300). (H) Pearson’s Coefficient analysis indicating the level of colocalisation of a MT-Qsox1::FLAG::HA construct transfected into S2R+ cells visualized with an HA antibody and antibodies against markers for the ER (Cnx99a), the Golgi (Golgin 84, Golgin 245, and GMAP), the early endosome (Hrs), the late endosome (Rab7) and the nucleus (DAPI) (n = 11–15) as well as with a srpHemo-mrva::3xmCherry construct (n = 18). (I) Western blot of concentrated supernatant collected from S2R+ cells transfected with srpGal4 UAS-qsox1::FLAG::HA (first three lines) and S2R+ cells that are untransfected. (J) Quantification of intracellular LanA intensity along a 4 μm line in macrophages (as indicated in schematic) from the control (black), minerva3102 (blue) and the qsox1KG04615 mutants (orange) (n = 4–5 embryos, 80–100 cells, 240–300 lines). For the whole graph see Figure 5—figure supplement 1G–J. Scale bars 50 μm for A, 30 μm in D. B-C, E-G and J were analyzed with Student’s test. ns = p > 0.05, *p<0.05, **p<0.01, ***p<0.001. See also Figure 5—figure supplement 1.

Figure 5—source data 1. Source data on the quantification of macrophages in the germband shown in Figure 5B-C, on the yolk shown in Figure 5—figure supplement 1A,1D, on the vnc shown in Figure 5—figure supplement 1B,1E, and in the whole embryo shown in Figure 5—figure supplement 1C.
Source data on the xyz position of macrophages from the tracks that form the basis of the analysis shown in Figure 5E-G. Source data on the quantification of the Pearson's coefficient for Qsox1 colocalization with different markers shown in Figure 5H and the quantification of LanA intensity shown in Figure 5J and Figure 5—figure supplement 1L-N. Source data on the xyz position of macrophages in the movies of the qsox1KG04615 mutant underlying the analysis shown in Figure 5E-G and Figure 5—figure supplement 1F.
elife-41801-fig5-data1.xlsx (1.9MB, xlsx)
DOI: 10.7554/eLife.41801.020

Figure 5.

Figure 5—figure supplement 1. Qsox1 affects germband entry and Laminin A.

Figure 5—figure supplement 1.

(A–B) Quantification of macrophages in fixed mid-Stage 12 embryos in control and qsox1KG04615 mutants reveals that in the mutant there are (A) increased numbers of macrophages on the yolk neighboring the germband (n = 18, p=0.002) and (B) reduced numbers on some vnc segments (n = 21; p value T1 = 0.357, T2 = 0.0006, T3 = 0.031, A1 = 0.034, A2 = 0.329). (C) Quantification of macrophages in the whole embryo in the qsox1 KG04615 mutant compared to the control (n = 12–14, p value=0.999) shows no change. (D–E) Quantification of macrophages in fixed mid-Stage 12 embryos from the control and upon knockdown in macrophages of Qsox1 with RNAi (v108288) driven by srpHemo-GAL4 reveals that in the mutant there are (D) increased numbers of macrophages on the yolk neighboring the germband (n = 23,24, p=0.02) and (E) reduced numbers on some vnc segments (n = 13–15; p value T1 = 0.024, T2 = 0.030, T3 = 0.258, A1 = 0.445, A2 = 0.233). (F) Analysis of the persistence of macrophages in the head in the qsox1KG04615 mutant compared to the control shows no significant difference (n = 3 movies for each, p=0.126). (G–J) Qsox1::FLAG::HA produced from an MT promoter visualized by HA-antibody staining (green) with (G–I) different parts of the endomembrane system as indicated visualized by antibody staining (green) and (J) Mrva::3xmCherry from the srpHemo promoter visualized by mCherry fluorescence. (K) Confocal images of LanA (green) staining in control, mrva3102 and qsox1KG04615 mutant embryos expressing cytoplasmic 3xmCherry (red) in macrophages from the srpHemo promoter. Dotted white lines in the LanA channel correspond to an outline traced from the mCherry channel. (L–N) Quantification of LanA and mCherry (red) intensity along a 4 μm line in macrophages from control (black line), mrva3102 (blue) and qsox1KG04615 mutant (orange) (n = 4–5 embryos, 80–100 cells, 240–300 lines). (M–N) Magnified view of LanA quantification from (M) the cell edge and (N) outside the cell. Scale bar is 5 μm in G-K. A-F and L-N were analyzed with Student’s t test. ns = p > 0.05, *p<0.05, **p<0.01, ***p<0.001.
Figure 5—video 1. Representative movie of macrophage migration into the germband in the qsox1KG04615 mutant.
Download video file (4.6MB, mp4)
DOI: 10.7554/eLife.41801.021
Macrophages (red) are labeled with srpHemo-H2A::3xmCherry. The time interval between each acquisition is 40 s and the display rate is 15 frames/sec. Scale bar represents 30 μm.