Septate Junction Proteins Play Essential Roles in Morphogenesis Throughout Embryonic Development in Drosophila

Abstract

The septate junction (SJ) is the occluding junction found in the ectodermal epithelia of invertebrate organisms, and is essential to maintain chemically distinct compartments in epithelial organs, to provide the blood-brain barrier in the nervous system, and to provide an important line of defense against invading pathogens. More than 20 genes have been identified to function in the establishment or maintenance of SJs in Drosophila melanogaster Numerous studies have demonstrated the cell biological function of these proteins in establishing the occluding junction, whereas very few studies have examined further developmental roles for them. Here we examined embryos with mutations in nine different core SJ genes and found that all nine result in defects in embryonic development as early as germ band retraction, with the most penetrant defect observed in head involution. SJ genes are also required for cell shape changes and cell rearrangements that drive the elongation of the salivary gland during midembryogenesis. Interestingly, these developmental events occur at a time prior to the formation of the occluding junction, when SJ proteins localize along the lateral membrane and have not yet coalesced into the region of the SJ. Together, these observations reveal an underappreciated role for a large group of SJ genes in essential developmental events during embryogenesis, and suggest that the function of these proteins in facilitating cell shape changes and rearrangements is independent of their role in the occluding junction.

Keywords: dorsal closure; head involution; morphogenesis; salivary glands; septate junction.

Figure 1

Loss of function mutations in SJ genes result in embryonic lethality with defects in head involution and dorsal closure. DIC photomicrographs (A–F, H–L) and a brightfield photomicrograph (G) of cuticle preparations of w¹¹¹⁸ (A and J), Cont^ex956 (B and F), Tsf2^KG01571 (C), Nrx-IV⁴³⁰⁴ (D), Nrg¹⁷ (E and G–I), Mcr¹ (K), and kune^c309 (L). Anterior is to the left and dorsal is up or facing. Defects in dorsal closure are indicated by cuticular scabs or holes on the dorsal surface (asterisks in B–D), whereas defects in head involution vary from completely uninvoluted head structures (arrows in E and F), to incomplete head involution where head skeletal structures are compressed anteriorly (arrow in K), to underdeveloped head skeleton (arrow in L points to a head skeleton lacking most of the dorsal and ventral processes). A number of embryos showed an “Empty Cuticle” phenotype (G), that when devitellinized varied from having a thin cuticle barely capable of holding the embryo together (H), to little or no cuticle resulting in disorganized mass of cells (I). Note the cuticle has separated from the epidermis in all of the mutant animals (small black arrows), but not in the w¹¹¹⁸ animals. Scale bars = 100 μm in A–F, H, and I, 60 μm in G, and 50 μm in J–L.

Figure 2

Mutations in SJ genes result in early arrested embryos. (A) Confocal optical section of a field of embryos 17–18 hr after egg laying from a self-cross of cor⁴/CyO, Dfd-YFP adults stained with antibodies against α-Spectrin (red and in channel A’) and with DAPI (blue and in channel A”). Note that four of the embryos have secreted cuticle and are refractory to antibody staining, whereas the second embryo from the left is a cor⁴ homozygous mutant (identified by absence of YFP, not shown) that has not secreted cuticle and can be stained. (B–D) Confocal optical sections of Cont^ex956 (B), kune^c309 (C), and Mcr^EY07421 (D) mutant embryos aged 17–18 hr after egg laying and stained with antibodies against α-Spectrin to show the general morphology of the embryo. Anterior is to the left. Note that the Cont embryo has arrested prior to the initiation of germ band retraction, the kune embryo is arrested at late stage 14/early stage 15, and the Mcr mutant embryo is generally disorganized. (E) Quantitation of developmental stages of w¹¹¹⁸ and SJ mutant embryos from a 1 hr embryo collection, subsequently aged 17 hr and then stained with antibodies against α-Spectrin. Note that all 10 SJ mutants had a significantly higher percentage of embryos arrested prior to stage 15 than the w¹¹¹⁸ control (Fisher’s exact test). n, total numbers of embryos scored for each genotype. * P < 0.05, ** P < 0.0001. Scale bars = 100 μm.

Figure 3

SJ proteins are expressed as early as stage 10 of embryogenesis. Confocal optical sections of stage 10–11 w¹¹¹⁸ embryos stained with antibodies against Cont (A), Cor (B), Kune (C), Mcr (D), Nrg (E), and Nrx-IV (F). Scale bar = 100 μm.

Figure 4

SJ mutant SGs exhibit abnormal morphology. Single confocal optical section selected from z-stack that revealed the largest number of cells in cross-section from stage 16 w¹¹¹⁸ (A) Cont^ex956 (B), kun^c309 (C), Mcr¹ (D), Nrg¹⁴ (E), and fkh-GAL4 > Mcr-RNAi (F) SGs stained with antibodies against ATPα (red, and in individual channels in center panels) to outline cells and against Uif (green, and in individual channels at far right) to mark the apical membrane, and with DAPI (blue) to mark the nuclei. Note that many of the SJ mutant SGs are shorter and fatter than w¹¹¹⁸, and have a broad lumen. The Nrg¹⁴ SG (E) is most similar to wild type. Scale bars = 20 μm.

Figure 5

Developmental time course of SG organogenesis from wild-type, Cont^ex956, and Mcr¹ mutant embryos reveals defective cell rearrangements in the mutant glands. Confocal z-series rendered in xz transverse cross-section of stage 11–16 w¹¹¹⁸ (A–D), Cont^ex956 (E–H), and Mcr¹ (I–L) SGs stained with antibodies against Uif to mark the apical membrane and Hoescht to label nuclei. The basal surface of each SG is indicated by the yellow line. Note that the number of nuclei surrounding the lumen in w¹¹¹⁸ SGs decreases from stage 11 to stage 16, whereas the number of nuclei surrounding the lumen of SJ glands does not. These views also highlight the broad, unexpanded or asymmetric lumen found in the mutant glands (arrows in G, H, K and L). Scale bars = 100 pixels.

Figure 6

SJ biogenesis and its relationship to embryonic morphogenesis. Comparison of developmental events (top row) to SJ protein localization (second row), along with timing of SJ ultrastructure [as determined by Tepass and Hartenstein (1994)] (third row) and timing of the occluding function of the junction [as determined by Paul et al. (2003)] (bottom row). The top row shows fluorescence photomicrographs of w¹¹¹⁸ embryos stained with FITC-labeled antiphosphotyrosine antibodies (PY). Anterior is to the left and dorsal is up in stages 12–14 and facing in stages 15 and 16/17. Scale bar = 100 μm. Second row shows confocal optical sections of SGs from stage 12 to 16 w¹¹¹⁸ embryos stained with antibodies against Cor. Scale bar = 20 μm. Note that all major developmental events are occurring and are mostly complete before the SJ is organized and physiologically tight. See text for details. DC, dorsal closure; HI, head involution.