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. 2017 Jun 22;6:e18481. doi: 10.7554/eLife.18481

Figure 5. Slit/Robo signalling and MMP2 control asymmetry of L/R phrenic nerves.

(A) NF staining of E14.5 diaphragm from Robo1+/+ and Robo2+/+ and Robo1–/– and Robo2–/–- embryos, left and right primary branches are pseudocolored in green and red,respectively, and superimposed to show the lack of asymmetry in the Robo1 and 2–/– embryos. Histogram showing the branch number and the defasciculation distance in Robo1+/+ and Robo2+/+, Robo1+/– and Robo2+/– and Robo1–/– and Robo2–/– embryos (R/L branch ratio: Robo1+/+ and Robo2+/+ 2.30 ± 0.37, versus Robo1–/– and Robo2–/– 1.06 ± 0.06; p=0.00048; R/L distance ratio: Robo1+/+ and Robo2+/+ 4.99 ± 0.89, versus Robo1–/– and Robo2–/– 1.05 ± 0.07; p=3E-6, Mann-Whitney). (B) Immunodetection of Robo1 and loading control (Tub) in left and right HB9::GFP ventral cervical spinal cord and distribution of the relative amount of the two shorter forms (pink arrowheads) to the full-length form (black arrowhead). The graph shows the normalized left and right values obtained for the five western-blots (dots, 6–8 embryos per sample) and the mean ± SEM (R versus L: p=0.01587, Wilcoxon singed rank); average fold-change is shown in brackets (1.22 ± 0.10). Normalization between lines was done on the Robo1 long form. (C) Ladder graph showing the left and right expression of Mmp2 detected by microarray in three embryos. Average Log2(R/L ratio) shown in brackets. (D) Photomicrograph of cultured ventral cervical spinal cord motoneuron. The combination of in situ zymmography with DQ-Gelatin and Islet1/2 staining enables the identification of motoneuron with MMP gelatinase activity. Histogram showing the amount of motoneuron with gelatinase activity in left and right samples (left 23.37% ± 2.7, N = 792 versus right 37.94% ± 2.1, N = 797; p=0.00109, Mann-Whitney). Histogram showing the gelatinase activity measured in cultures from Rfx3–/– embryos with symmetric lungs (Iso) and in cultures from Rfx3+/+, Rfx3+/– embryos (Rfx3 wt: — 1.4 ± 0.08; Rfx3 iso — 0.96 ± 0.08, p=0.0013, Mann-Whitney). (E) NF staining of E14.5 diaphragms from wild-type and Mmp2–/– embryos. Left (green) and right (red) primary and secondary branch traces shown in the middle panel are superimposed in the right panel to compare the left and right patterns. Histograms showing the R/L ratios of branch number and defasciculation distances. Ratio of secondary branches: Mmp2+/+ and Mmp2–/+ 1.74 ± 0.07, versus Mmp2–/– 1.21 ± 0.10; p=0.00029; defasciculation distance: Mmp2+/+ and Mmp2–/+ 5.33 ± 0.44, versus Mmp2–/– 3.49 ± 0.38; p=0.022, Mann-Whitney. Scale bar: 200 μm (A,E), 10 μm (D). Numerical values used to generate the graphs are accessible in Figure 5—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.18481.022

Figure 5—source data 1. Slit/Robo signalling controls asymmetry of L/R phrenic nerves and Robo1 exhibits different processing levels in left and right cervical motoneurons.
This file provides the statistical report and individual values used to create the histograms and ladder graphs shown in Figure 5A, B, C, D and E. The ratios of branch numbers and defasciculation distances in Robo1+/+ and Robo2+/+, Robo1+/– and Robo2+/– and Robo1–/– and Robo2–/– embryos are shown on the first and second sheets. The third sheet contains left and right normalized values of short Robo1 forms presented in the graph of Figure 5B. RNA levels of Mmp2 are shown on fourth sheet. The percentage of left and right motoneurons (Islet+) exhibiting gelatinase activity from wild-type embryos are shown on fifth sheet and the ratio found in Rfx3–/– embryos with lung isomerism and Rfx3+/+ and Rfx3+/– on the sixth sheet. Branch number and defasciculation distance ratio measured in Mmp2+/+ and Mmp2–/– embryos are shown on the seventh and eighth sheets.
DOI: http://dx.doi.org/10.7554/eLife.18481.023
elife-18481-fig5-data1.xlsx (85.9KB, xlsx)
DOI: 10.7554/eLife.18481.023

Figure 5.

Figure 5—figure supplement 1. Post-translational regulation of Robo1.

Figure 5—figure supplement 1.

(A) Ladder graph of Robo1 RNA expression detected by the two probes present on the microarray for each of the three embryos (probe1 — black; probe2 — blue; average Log2(R/L ratio) shown in brackets). (B) Histogram showing the average R/L ratio of expression (in log2) assessed by the microarray probes targeting Slit1, Slit2 and Slit3. Error bars represent SEM. (Note that 1.5 fold-change gives 0.5849 in log2.) (C) Immunodetection of Robo1 in spinal cord lysates of Robo1–/– and Robo2–/– and wild-type tissues. The antibody detects three specific bands. Black arrowhead points to the expected full-length Robo1 and the two pink arrowheads point to the two shorter forms. (D) Graph shows the normalized left and right values obtained for the four western-blots (6–8 embryos per sample) and the mean ± SEM (R versus L: p=0.028, Mann-Whitney; average fold-change is 1.22 ± 0.11). Normalization between lines was done on the tubulin band. Numerical values used to generate the graphs are accessible in Figure 5—figure supplement 1—source data 1.
DOI: http://dx.doi.org/10.7554/eLife.18481.024
Figure 5—figure supplement 1—source data 1. Post-translational regulation of Robo1 and biased expression of Mmp2.
This file provides the statistical report and individual values used to create the histograms and ladder graphs shown in Figure 5—figure supplement 1A and B. Left and right levels or ratios for the Robo1 and the Slits transcripts are shown on the first and second sheet, respectively. The third sheet contains left and right tubulin normalized values of short Robo1 forms presented in the graph of Figure 5—figure supplement 1D.
DOI: http://dx.doi.org/10.7554/eLife.18481.025
elife-18481-fig5-figsupp1-data1.xlsx (73.3KB, xlsx)
DOI: 10.7554/eLife.18481.025

Figure 5—figure supplement 2. Asymmetric expression of MMP2.

Figure 5—figure supplement 2.

(A) Histogram showing the average ratio of Mmp2 RNA expression assessed by qPCR in cervical motoneurons from two E11 embryos. Expression normalized to GAPDH. (B) Histogram showing the R/L ratio (in log2) of the surface labelled by the RNAscope MMP2 probe in the motoneuron region. Each bar shows the R/L ratio for one section of a series of serial sections that cover the entire cervical spinal cord region. The dashed line highlights the log2 value that corresponds to a 1.5-fold change. (C) Mmp2 RNAscope in situ hybridization on an E12 transversal spinal cord section at cervical levels. (D) Mmp2 in situ hybridization combined with Pou3f1 immunolabeling on E11 transversal spinal cord sections. Enlarged panels of the motoneuron domain (right) show that Mmp2 transcripts are detected within the Pou3f1-positive domain. (E) Schematics of the dissection and the in situ zymography (ISZ) procedure for E12.5 ventral cervical spinal cord. Cleavage-induced fluorescence of DQ-Gelatin (green) is overlaid over the phase contrast image. Islet1-positive motoneurons exhibit gelatinase activity in different cellular regions, including the axon and the growth cone (lower right panel). (F) Ladder graph showing the expression signals in the left and right laser-captured samples of the three embryos detected with the microarray Mmp14, Mmp15, Mmp16 and Mmp17 probes, average Log2(R/L ratio) shown in brackets. Scale bars: 100 μm (C) and (D left panel); 200 μm (D right panel) and 10 μm (E). Numerical values used to generate the graphs are accessible in Figure 5—figure supplement 2—source data 2.
DOI: http://dx.doi.org/10.7554/eLife.18481.026
Figure 5—figure supplement 2—source data 2. Asymmetric expression of Mmp2 in cervical motoneurons and expression of other MMPs.
Mmp2 expression ratios quantified by qRT-PCR and quantitative in situ hybridization (RNAscope) are presented on the first and second sheets, respectively. The third sheet shows the microarray data used to generate the ladder graphs shown in Figure 5—figure supplement 2F.
DOI: http://dx.doi.org/10.7554/eLife.18481.027
elife-18481-fig5-figsupp2-data2.xlsx (52.5KB, xlsx)
DOI: 10.7554/eLife.18481.027