Figure 1. Protein kinase A (PKA) is required for blood–brain barrier (BBB) formation and acts in the Moody signaling pathway.

(A) Single confocal sections of dye-injected embryos of WT and PKA zygotic mutants. (B) Quantification of the dye penetration assay. Columns represent the intensity of dye penetration into the nerve cord as measured by the mean pixel intensity (see Experimental procedures), ± SEM, n = 32, 31, 41, 38, 16 in WT, PkaC1^B3, PkaC1^H2, PkaC1^A13, Loco^∆13 embryos, respectively. Loco^∆13 zygotic mutants serve as positive controls. (C) Repo staining revealing the number and positions of subperineural glia (SPG) nuclei in WT and PKA zygotic mutants using an illuminated projection to highlight the ventral surface of the nerve cord. (D) Transmission electron micrographs of the interface of neighboring SPG in late WT and PkaC1^H2 zygotic mutant embryos. Yellow brackets delineate the septate junction (SJ) ultrastructure; high magnifications are shown in red boxes. (E) Quantification of SJ length in WT and PkaC1^H2 mutants (see Experimental procedures). Columns represent mean SJ length as measured in random nerve cord sections, ± SEM, n = 56 and n = 70 in WT and PkaC1^H2 mutants, respectively. (F) Time-lapse recording of BBB closure in embryos of WT and PKA zygotic mutants. 6 µm confocal stacks are shown; in each image, 4–6 ventral SPG are highlighted (green); midline channels (stars) and retarded growth (arrows) are marked. (G) Dominant genetic interactions between PkaC1^B3 and Gβ13F^∆1-96A as quantified by dye penetration in the embryo. Columns represent the intensity of dye penetration as measured by the mean pixel intensity, ± SEM, n = 34, n = 48, and n = 71 in PkaC1^B3/+, Gβ13F^∆1-96A/+, and Gβ13F^∆1-96A/+;PkaC1^B3/+ mutants, respectively. In (B) and (G), the percentage of embryos showing the dye penetration is indicated at the bottom of each column. Brackets and asterisks in (B), (E), and (G) indicate statistical significance levels as assessed by ordinary one-way ANOVA with Dunnett’s multiple comparisons test in (B) and (G) or the two-tailed Student’s t-test in (E), n.s., p>0.05; *p<0.05; **p<0.01; ***p<0.001.

Figure 1—figure supplement 1. Genetic interactions between protein kinase A and different Moody pathway components.

Quantitative analysis of dye penetration shows that the mild blood–brain barrier defect of PkaC1^B3 heterozygotes can be partially rescued by removing one copy of Gβ13F as well as one copy of Loco. The removal of one copy of other Moody pathway components, such as moody, Gαo, and Gαi, results in no or weaker, non-significant rescue, suggesting that Gβ13F is more dosage sensitive. Asterisks indicate statistical significance levels as assessed by one-way ANOVA with Dunnett’s multiple comparisons test, n.s., p>0.05; *p<0.05; **p<0.01; ***p<0.001.

Figure 1—video 1. Subperineural glia (SPG) epithelium formation in WT embryos.

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The SPG (labeled by the membrane marker GapGFP and the actin marker MoesinGFP, driven by the pan-glial driver repoGAL4) are fairly uniform in size and cell shape, and their spreading and cell–cell contact formation are highly synchronized. The glial sheet is closed by 15.5 hr of development.

Figure 1—video 2. Subperineural glia (SPG) epithelium formation in protein kinase A (PKA) zygotic null mutant embryos.

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The SPG of PkaC1^B3 zygotic mutants show variable size and cell shape, and their spreading and contact formation are poorly synchronized, resulting in patchy cell–cell contacts with gaps of different sizes. Moreover, the complete closure of the SPG epithelium is delayed compared to WT.