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

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© 2019, Valoskova et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 3. — (A–C) Representative confocal images of early Stage 12 embryos from (A) control, (B) P{EP}CG8602^EP3102 = minerva (mrva)³¹⁰² mutant, and (C) mrva³¹⁰² mutants with macrophage expression of the gene rescued by srpHemo(macro)-mrva. Macrophages express srpHemo-3XmCherry (red) and the embryo autofluoresces (green). In the mutant, macrophages remain in the head and fail to enter the germband, hence we name the gene minerva. (D) Dashed ellipse in schematic at left represents the germband region in which macrophages (red) were counted throughout the study. Comparison of the control (n= 38), mrva³¹⁰² mutants (n = 37) and mrva³¹⁰² mutant/Df(3L)BSC117 that removes the gene (n = 23) shows that the mutant significantly decreases migration into the extended germband (p<0.0001 for control vs mutant, p=-0002 for control vs Df cross). This defect can be partially rescued by expression in macrophages of srpHemo >mrva::FLAG::HA (n = 18, p=0.222 for control vs rescue, p=0.036 for mutant vs rescue) and completely rescued by precise excision (mrva^Δ32) of the P element (n = 16, p=0.826). srpHemo >mCherry nls labeled the macrophages. (E–G) Macrophage quantification in early Stage 12 embryos. (E) Fewer germband macrophages upon expression of mrva RNAi v101575 only in macrophages under the control of srpHemo (n = 28–35 embryos, p<0.0001). (F) Fewer macrophages found on the yolk neighboring the germband (oval in schematic) in the mrva³¹⁰² mutant compared to control embryos (n = 14–16 embryos, p=0.0003). (G) Increased germband macrophage numbers in shg^P34; mrva³¹⁰² compared to the mrva³¹⁰² mutant indicates a partial rescue from reducing DE-Cadherin which is expressed in the germband ectoderm (n = 19–29, p<0.0001, p=0.005). (H) No significant difference in number of macrophages labeled with srpHemo-3xmCherry in vnc segments (area in blue oval in schematic) between control and mrva³¹⁰² mutant embryos in fixed mid Stage 12 embryos (n = 23–25, p=0.55). Images from two-photon movies of (I) Stage 10 and (L) late Stage 11-early Stage 12 embryos in which macrophage nuclei (red) are labeled with srpHemo-H2A::3xmCherry. (I) Stills at 0 and 60 min and (J) quantification of macrophage speed reveal 33% slower macrophage migration in the head towards the yolk neighboring the germband in the mrva³¹⁰² mutant compared to the control, n = 3 movies for each, #tracks: control = 329, mutant = 340, p=0.002. Blue box in magnification in schematic indicates region analysed in J. (K) The first macrophage in mrva³¹⁰² mutants is much slower to enter the germband after macrophages reach the germband edge (control = 22.00 ± 1.53 min, n = 3, mrva³¹⁰² mutant = 102.0 ± 20.35 min, n = 4. p-value=0.021). (L) The time when macrophages reached the germband in each genotype was defined as 0’. Stills at 60 and 90 min and (M) quantification of macrophage speed reveal 43% slower macrophage migration in the germband in the mrva³¹⁰² mutant compared to the control. Blue arrow in schematic indicates route analyzed. n = 3 movies for each, #tracks: control = 21, mutant = 14, p=0.022. Significance was assessed by Kruskal-Wallis test with Conover post test comparison in D, G, Student’s t-test in E, F, H, J-K, M. ns = p > 0.05, *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. Scale bars are 50 μm in A-C, 40 μm in I, 30 μm in L. See also Figure 3—figure supplement 1 and Figure 3—video 1–3.

Figure 3—source data 1. Source data on the quantification of macrophages in the germband shown in Figure 3D–E,G and Figure 3—figure supplement 1A, on the yolk (Figure 3F) on the vnc (Figure 3H, Figure 3—figure supplement 1C) and in the whole embryo (Figure 3—figure supplement 1D–E).
Source data on the xyz position of macrophages from the tracks that form the basis of the analysis shown in Figure 3J–K,M and Figure 3—figure supplement 1F,G. Source data on the mismigration of germ cells, the levels of mrva expression in the ovary, and the migration of border cells (Figure 3—figure supplement 1I,J and L, respectively). Source data on the xyz position of macrophages in the movies of the mrva³¹⁰² mutant and the control underlying the analysis shown in Figure 3J-K,M and Figure 3—figure supplement 1F-G.

elife-41801-fig3-data1.xlsx^{(3.5MB, xlsx)}

DOI: 10.7554/eLife.41801.011

Figure 3—figure supplement 1. — (A–C) Representative confocal images of early Stage 12 embryos from (A) control, (B) P{EP}CG8602^EP3102 = minerva (mrva)³¹⁰² mutant, and (C) mrva³¹⁰² mutants with macrophage expression of the gene rescued by srpHemo(macro)-mrva. Macrophages express srpHemo-3XmCherry (red) and the embryo autofluoresces (green). In the mutant, macrophages remain in the head and fail to enter the germband, hence we name the gene minerva. (D) Dashed ellipse in schematic at left represents the germband region in which macrophages (red) were counted throughout the study. Comparison of the control (n= 38), mrva³¹⁰² mutants (n = 37) and mrva³¹⁰² mutant/Df(3L)BSC117 that removes the gene (n = 23) shows that the mutant significantly decreases migration into the extended germband (p<0.0001 for control vs mutant, p=-0002 for control vs Df cross). This defect can be partially rescued by expression in macrophages of srpHemo >mrva::FLAG::HA (n = 18, p=0.222 for control vs rescue, p=0.036 for mutant vs rescue) and completely rescued by precise excision (mrva^Δ32) of the P element (n = 16, p=0.826). srpHemo >mCherry nls labeled the macrophages. (E–G) Macrophage quantification in early Stage 12 embryos. (E) Fewer germband macrophages upon expression of mrva RNAi v101575 only in macrophages under the control of srpHemo (n = 28–35 embryos, p<0.0001). (F) Fewer macrophages found on the yolk neighboring the germband (oval in schematic) in the mrva³¹⁰² mutant compared to control embryos (n = 14–16 embryos, p=0.0003). (G) Increased germband macrophage numbers in shg^P34; mrva³¹⁰² compared to the mrva³¹⁰² mutant indicates a partial rescue from reducing DE-Cadherin which is expressed in the germband ectoderm (n = 19–29, p<0.0001, p=0.005). (H) No significant difference in number of macrophages labeled with srpHemo-3xmCherry in vnc segments (area in blue oval in schematic) between control and mrva³¹⁰² mutant embryos in fixed mid Stage 12 embryos (n = 23–25, p=0.55). Images from two-photon movies of (I) Stage 10 and (L) late Stage 11-early Stage 12 embryos in which macrophage nuclei (red) are labeled with srpHemo-H2A::3xmCherry. (I) Stills at 0 and 60 min and (J) quantification of macrophage speed reveal 33% slower macrophage migration in the head towards the yolk neighboring the germband in the mrva³¹⁰² mutant compared to the control, n = 3 movies for each, #tracks: control = 329, mutant = 340, p=0.002. Blue box in magnification in schematic indicates region analysed in J. (K) The first macrophage in mrva³¹⁰² mutants is much slower to enter the germband after macrophages reach the germband edge (control = 22.00 ± 1.53 min, n = 3, mrva³¹⁰² mutant = 102.0 ± 20.35 min, n = 4. p-value=0.021). (L) The time when macrophages reached the germband in each genotype was defined as 0’. Stills at 60 and 90 min and (M) quantification of macrophage speed reveal 43% slower macrophage migration in the germband in the mrva³¹⁰² mutant compared to the control. Blue arrow in schematic indicates route analyzed. n = 3 movies for each, #tracks: control = 21, mutant = 14, p=0.022. Significance was assessed by Kruskal-Wallis test with Conover post test comparison in D, G, Student’s t-test in E, F, H, J-K, M. ns = p > 0.05, *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. Scale bars are 50 μm in A-C, 40 μm in I, 30 μm in L. See also Figure 3—figure supplement 1 and Figure 3—video 1–3.

Figure 3—source data 1. Source data on the quantification of macrophages in the germband shown in Figure 3D–E,G and Figure 3—figure supplement 1A, on the yolk (Figure 3F) on the vnc (Figure 3H, Figure 3—figure supplement 1C) and in the whole embryo (Figure 3—figure supplement 1D–E).
Source data on the xyz position of macrophages from the tracks that form the basis of the analysis shown in Figure 3J–K,M and Figure 3—figure supplement 1F,G. Source data on the mismigration of germ cells, the levels of mrva expression in the ovary, and the migration of border cells (Figure 3—figure supplement 1I,J and L, respectively). Source data on the xyz position of macrophages in the movies of the mrva³¹⁰² mutant and the control underlying the analysis shown in Figure 3J-K,M and Figure 3—figure supplement 1F-G.

elife-41801-fig3-data1.xlsx^{(3.5MB, xlsx)}

DOI: 10.7554/eLife.41801.011